Erwin Knecht—the intelligent and mad, funny and grumpy man of autophagy

ABSTRACT

Getting to know Erwin Knecht is not so simple. This view was summarized in a comment from Dr. Marta Martinez Vicente, who obtained her PhD degree working in a lab that shared space with Erwin’s group: “Erwin Knecht is a complex character, who awakens contradictory feelings. To define him I would say that he is a mixture of intelligence and madness, he’s witty, very funny but also grumpy and cranky, all mixed and all simultaneously. Without a doubt he is a person who will not leave anyone indifferent, his original personality marked all who crossed his path, doctoral students, collaborators, people who attended his talks, etc … I remember the weekly lab meetings with him; we, the students, had a lot of respect for him (not to mention fear), but his comments were always smart, helpful and constructive, he has always been prone to helping everyone. In the lab, he was extremely demanding, but got people under his supervision to do their best. And despite his usual moodiness, he managed to make everyone love him and have a special affection for him. Above all, I remember his screams throughout the laboratory that could be heard from all over the building, calling out to his laboratory technician: ‘Asunción!’ They were like an old couple, arguing all day but they couldn’t be without each other, it was like watching a sitcom every day.” If you are intrigued, please read on.

Daniel J. Klionsky: We met at a conference many years ago. If I remember correctly, I found out that you were actually born in Germany (I think I asked you about your last name). However, I do not remember if you told me how you ended up in Spain.

Erwin Knecht (Figure 1): I was born in Valencia (Spain), almost four years after the end of the Second World War. My father was a German (from Pforzheim, in Baden-Württemberg) who came to Spain and my mother was Spanish (from Valencia, but she got the German nationality when she married my father). I got automatically the German nationality, because in those times in Spain the nationality of the father was what decided that of wife and sons, independently of where you were born. In fact, this was still so, for many years, at least for children, since my son, who was born in 1976 in Valencia, is German in spite of the fight of my Spanish wife with Spanish bureaucracy to include him in her Spanish passport. However, although I was living in Valencia and I refused the offer for going off to my German family, I did not see any reason to change my original nationality. Initially, when I was 18 years old and had to decide, I chose to be German, because I could avoid in this way the military service as a soldier in Spain and in particular in its colonies in Africa, and later, when I was a PhD, to be German was also okay, because German postdoctoral fellowships provided more money than Spanish fellowships (for example, in 1980 and for the USA my post-doctoral German fellowship was almost 3 times higher than a prestigious Spanish fellowship of my wife). And another reason to remain German in Spain was that I don’t like the queues and the difficulties from the Spanish bureaucracy. Now, Spain belongs to the European Community like Germany, so hopefully there will not be problems anymore with nationality. Therefore, I have remained a German with a German passport living in Spain (I still have part of my family in Germany, a brother, an aunt and various cousins) and will most probably die here without changing my nationality.

Figure 1.

Erwin Knecht, circa 2007. Photo courtesy of Ghita Ghislat.

DJK: One of the people who collaborated with you, Paul Saftig (Christian-Albrecht University of Kiel) had an interesting comment about your dual nationality: “Not only due to his German family name but also due to his half-German education it was fascinating to meet somebody with mixed cultures, i.e. on the one hand being relaxed and enjoying the ‘dolce vita’ and on the other hand the crazy German way to be critical, reserved and sometimes being too structured.”

EK: I would like to agree with Paul, since this probably would explain why I like to the same extent German beer and Spanish red wine. But this has also some disadvantages because I was educated in a way to be almost a fanatic of punctuality (die deutsche Pünktlichkeit) and this precisely is not a virtue here in Spain and has resulted in many arguments and problems with certain people. On the Spanish influence, I remember that I was talking at a party with Prof. Helmut Holzer from the Universität Freiburg and noticed that he didn’t remember me, so I tried to tell him my name and asked him: Do you remember me? But he, who obviously didn’t remember me but was an old fox, answered: “How can I forget your strong Spanish accent?”

DJK: Can you tell me about your educational background? Where did you attend university, and graduate school? Postdoc? Who did you work with, and on what topics?

EK: I was educated in Valencia, the third largest city of Spain (after Madrid and Barcelona), which is situated at the Mediterranean Sea and, because there were many German traders working here, there was, since 1909, a German School that I attended. I learned Biological Sciences at the University of Valencia because, although the school advisors oriented me to mathematics and I took a first year in engineering, I was much more interested in cell biology research, so I decided to change my studies to biology. Then, once I graduated in June 1972, I was lucky enough to obtain in September of that year, with two other colleagues and among other candidates after three days of examinations, a competitive fellowship at the laboratory of Electron Microscopy of a private institution, the Instituto de Investigaciones Citológicas (IIC). I say I was lucky because these were the last years of the dictatorship of general Franco, a residual survivor of the fascist leaders in Europe that were removed from their chairs when the allies won the Second World War in 1945. As a consequence of its political isolation from the democratic countries (the period of Spanish autocracy), Spain was not rich and science was underdeveloped, since its government had other priorities. Therefore, fellowships to carry out scientific research to get a PhD were very scarce here and, in addition, I, as a foreigner, could not apply at these times for an official position in Spain at a public school, university or research institution.

The IIC, in contrast, was a quite odd institution, because it was a private research institute entirely supported by a bank (Caja de Ahorros de Valencia). Savings banks like that one, were obligated by law in Spain to dedicate yearly part of its benefits to social activities, and the first IIC director, Prof. Gerónimo Forteza, was able, somehow (he probably was the personal doctor of one or more of the bankers, but I really don’t know), to persuade them to construct, with these social funds, a new building in the city for an institute of basic research and to maintain it, including the three above-mentioned fellowships and the staff salaries. Prof. Forteza was a fine hematologist interested in and working on microscopy procedures and, therefore and to his indications, the new research institute was very well equipped (much more, for example, than the University) for light and electron microscopy research and also for cell culture.

Here I learned, with the help of the director of the laboratory of electron microscopy, Dr. José Hernández-Yago, who was a real expert in these techniques that he mastered in different countries, and of other members of the IIC, but also by myself, all the basic electron microscopy procedures available at those times (physical and chemical fixation, embedding and ultrathin sectioning, transmission electron microscopy, electron microscopic cytochemistry and autoradiography, rotary shadowing, negative staining, scanning electron microscopy, freeze-etching and freeze-fracturing, morphometry and stereology, etc.). In fact, we organized during 1–2 weeks yearly and for thirty years a special private course of Techniques in Electron Microscopy for about 20–30 post-graduates from different Spanish universities, hospitals and research centers. With such a specialized technology, which in my opinion was a bit limited to ask modern relevant biological questions, I started the research for my PhD dissertation with full freedom, but with the necessary condition that it had to be mainly based in electron microscopy. Thus, when I obtained in 1972 my first ultrathin sections from a monkey kidney epithelium cultured cell line, I was immediately interested in lysosomes and related particles, because, due to their high morphological heterogeneity, they appeared to me much more interesting, of course from an electron microscopy point of view, than other cell organelles with a quite homogeneous morphology, like the nucleus, the ER, the Golgi complex or the mitochondria.

Knowing that these heterogeneous populations were functionally related, I decided for my doctoral thesis, first to organize all this group of particles in a number of classes on the basis of morphological criteria and next to associate to each a specific lysosomal-related function. So, and according to what I saw in the electron microscope, I divided first all these complex entities into classes (initially, about 20!) on the basis of the observed combinations of four morphological parameters: size (small/medium/large), number of membranes (one/two/four), matrix density (clear/medium/dense) and content (nothing/vesicles/identifiable cell components/membrane debris/amorphous material). Then, I tried to associate each morphological class to a specific lysosomal-related function, using different electron microscopy procedures (Figure 2). For example, to separate lysosomes from pre-lysosomes (hetero- and autophagosomes) I used electron microscopic cytochemistry of two lysosomal enzymes, acid phosphatase and aryl sulphatase, and to separate peroxisomes from lysosomes and pre-lysosomes I used a cytochemistry of catalase (being always careful enough with the incubation times so that the electron-dense precipitate formed in the cytochemical reaction did not hide the morphological structure of the organelle to allow its identification). To distinguish heterophagy (endocytosis) from autophagy, I incubated the cultured cells with colloidal silver and followed kinetically the incorporation of this dense marker from the plasma membrane into the different morphological structures of the cell at different times. Also, I carried out kinetic experiments in the opposite direction, i.e., from the Golgi complex to the periphery of the cell, using electron microscopic radioautography in pulse-chase experiments with ³H-fucose, to follow how labeled glycoproteins are incorporated along time from the Golgi complex into the specific morphological entities that I classified first. Finally, since the more or less dense lysosomes frequently show a typical clear “halo” inside their membranes, probably corresponding to the sugars of the lysosomal membrane glycoproteins and that we and others thought probably protected them against attack by the acid hydrolases, I used also a general cytochemical procedure for the localization of polysaccharides by electron microscopy. This allowed the identification of those morphological classes that, similar to plasma membranes, have a high content of mucopolysaccharides and glycoproteins on their membranes, in comparison with other cell membranes that cytochemically remained almost unlabeled, like nuclear or mitochondrial membranes.

Figure 2.

Erwin obtaining carbon films for electron microscopy in Valencia, circa 1973. Photo courtesy of Asunción Montaner.

These experiments allowed also the identification of other characteristics of some of the morphological classes. Thus, for example, glycogen, which obviously was also labeled in the cytochemical experiments to detect polysaccharides, was apparently taken up by lysosomes, both through autophagy accompanying the sequestered cell organelles (that corresponds to what was later called macroautophagy), but also through infolding and vesiculation of the lysosomal surface in multivesicular and in dense bodies (probably microautophagy). In summary, these experiments allowed me to combine all these results and to kinetically and functionally relate all these defined morphological structures in the monkey kidney epithelial cells, assigning to each class, once I separated the lipid droplets and the peroxisomes, a unique specific function: autophagosomes, autolysosomes, heterophagosomes, heterolysosomes, ambilysosomes (today called, I think, amphisomes), residual bodies and primary lysosomes, as shown in the last figure of the paper that I published later in 1978 (see below). I think that these studies are a modest example of the kind of work, in the field that we are commenting on here, at those early times, which were mostly based on electron microscopy procedures, since this was the main experimental tool then available to visualize and to investigate autophagy. In fact, and before 1976, there were few researchers working in autophagy, but certainly doing a much better job than me, since they quickly and continuously published their work. For example, working in autophagy or related areas, I remember of course Christian de Duve and his group, but also Benjamin F. Trump, Alex B. Novikoff, Ullrich Pfeifer, Arvid B. Maunsbach, János Kovács, Keith R. Porter, Kurt von Figura and Jan L.E. Ericsson, among others.

In any case, these studies with the monkey kidney cultured cells have been later quite useful for me to quickly and easily identify morphologically specific pre-lysosomal and lysosomal functional populations at the electron microscope and to determine, in combination with morphometric analyses, which ones were specifically affected by different treatments or conditions. With these studies, I presented the written account of my work (in Spanish) at the University of Valencia and, after the corresponding public dissertation before five examiners, members of the University, I received my PhD in 1975. At the same time, I got a contract as an associate researcher at the IIC. A few weeks later, the first IIC director unfortunately died and this same year (and even the same month I think) the dictator Franco finally died too, allowing Spain to be converted into a democracy in much better relations with European and American democratic countries and facilitating more in this way our scientific interactions. In one or two years, a new director for the Institute was appointed, Prof. Santiago Grisolía, an MD born in Valencia, but that migrated to the USA in 1949. The bank offered him quite favorable conditions and, thus, he finally left the University of Kansas, where he was Professor of Biochemistry, to return to Valencia as the new director of the IIC until 1991. I am particularly grateful to him, from who I learned many things, good and bad, about the scientific game, because he always kindly supported my ideas and my work and allowed me to do it quite independently.

In the beginning at the IIC there was no rush to publish, which I liked very much, but, finally, at the indications of the new director, I ended translating my PhD dissertation into English, and with no additional results to those already present in the original thesis, I submitted it for publication as a single paper in 1978 to the German journal, Cell Tissue Research (because, by then, this journal, and Protoplasma for example, accepted quite long papers for review) [1]. I always thought that, due to my limited experience in writing papers, the title “Ultastructural localization of polysaccharides in the vacuolar system of an established cell line” was quite inadequate for the large amount of studies compiled in that work. Anyway, as a post-doc, I got some fellowships and permission from my Institution for specific stays abroad to learn and to become familiar with other procedures and other areas of research. For example, I stayed at the Institute of Pharmacology et Toxicologie in Toulouse in France for a couple of months in 1976, and at the Department of Biological Sciences and at the Medical School of Stanford University in 1980/1981, working, respectively, on electron microscopy of mitochondrial DNA in leukemia, circadian rhythms in Neurospora crassa or endogenous analogs of digitalis in guinea pigs. Although these were certainly interesting fields, then and now I found more interesting the research on intracellular protein degradation.

DJK: That is an interesting coincidence – I started graduate school at Stanford, in the Department of Biology, in 1980, so we must have overlapped for a short time. Who did you work with in that department?

EK: Well, I started at the end of 1980 with Prof. Dow O. Woodward at the Department of Biology, because he was coauthor of a Nature paper on the possibility of export of mitochondrial proteins to the microsomal fraction in Neurospora crassa. In fact, my proposal for the fellowship was based on that. However, when I arrived at Dow’s lab, he was not further interested in this topic and continued working in circadian rhythms of Neurospora. So, I started testing, during a few months, possible changes in the Neurospora circadian rhythm with different chemicals. Dow at that time was above all an excellent teacher. I attended some of his lessons and I learned with him of Thomas Kuhn, Karl Popper, Paul Feyerabend and others. Also, I remember of those times the interesting lectures of Prof. William Charles Demens in his course on Sleep and Dreams, related with circadian rhythms. However, I soon found boring the routine work on Neurospora and, after talking with Dow, who was very nice and with whom I maintained a very good relationship, I decided to change my interest against the recommendations of my German fellowship advisor, who, however, could not stop me. I learned of the interesting work on endorphins (endogenous morphins) done in the laboratory of Avram Goldstein at the Department of Pharmacology in the Medical School and another Jewish scientist, Prof. Sumner M. Kalman, a very nice person and an expert in drug assays, accepted me, with the help of Professor Grisolía, in his laboratory at the Department of Pharmacology, after he talked with Dow. There, I worked on the purification from human plasma of possible endogenous analogues of digitalis, using a bioassay with atria from guinea pigs to test the fractions purified by classical biochemical procedures. I used to arrive very early to work at Stanford to avoid the heavy traffic on the San Francisco bridges, because my wife was a post-doc at Berkeley and I was living there with her and our 4-year-old son, and at the scintillation counters room I met Arthur Kornberg from the nearby Biochemistry Department, who used to visit this room quite early, probably to directly see by himself and enjoy the pleasure and excitement of seeing as first the results of the work carried out the day before by his students. He was nice enough to invite me to visit his office and discuss with him about past, present and future scientific topics. Talking to Prof. Kornberg was a real pleasure, as was later the few scientific discussions I had with Prof. Severo Ochoa in Spain, because both of them had an outstanding open mind and a vast scientific knowledge.

DJK: What specifically led you into the autophagy field?

EK: As I told you, it was mainly an esthetical reason: I was attracted by the beautiful variety of appearances in the electron microscope of lysosomes, pre-lysosomes and post-lysosomes from a monkey kidney epithelial cell line. Since my main initial research concern was then on lysosomes in general, I was obviously also interested in autophagic vacuoles in particular (autophagosomes, autolysosomes and ambilysosomes) from the very beginning of my research work in 1972.

The new director of the IIC was investigating, among other topics, enzymes, metabolites and allosteric effectors of the urea cycle and one of his many interests was the degradation of the corresponding mitochondrial proteins. Two Spanish members of his group that returned with him from Kansas to the IIC, Chelo Guerri and Vicente Rubio, had already published a few papers on that, but then they worked in a different area (fetal alcohol syndrome, Chelo, and molecular structure of carbamoyl phosphate synthetase I and other enzymes, Vicente). In my case, I was very happy to start my work with the degradation of mitochondrial proteins, which I liked most, because this allowed me to use my electron microscopy expertise and, at the same time, to incorporate new perspectives and methodologies from biochemistry, molecular biology and genetics to my cell biology background. I remember from those times in particular the influential review papers on the subject of intracellular protein degradation in the Annual Review of Biochemistry from Robert T. Schimke and Alfred L. Goldberg [2–4] in the USA and the proceedings from the Symposia on Intracellular Protein Catabolism, organized in East Germany and published in a poorly known European journal from this country, Acta Biologica et Medica Germanica. Once more during those times, around the 1980s, there were few researchers specifically working in autophagy or related areas. I remember Klaus Hendil, Roger T. Dean, John Ballard, Nobuhiko Katunuma (an outstanding and unusual Japanese Professor: I remember to have drunk more beer in one night in Valencia with him than ever before, so his aldehyde dehydrogenase was probably okay; and also I remember to have been talking at the very beginning in a meeting, I think in Germany, with Eiki Kominami and that he came to us and told Eiki indicating to me: He is your enemy), R. John Mayer, Martin Rechsteiner, Fred Meijer and Hans Glaumann, among others. But more especially, there were the works on autophagy of Per O. Seglen in Norway with his hepatocyte cultures and Glenn E. Mortimore in Pennsylvania with his amino acid studies.

However, I sometimes doubted if lysosomal protein degradation was a good choice for my future research, since until this century I have been always criticized by other colleagues for working on something like a garbage dump instead of choosing more fashionable research areas at those times such as protein synthesis or DNA replication. My former wife for example, Maria Eugenia Armengod, worked at that time in genetic recombination with Escherichia coli. I remember that during a visit to a professor in Gif-Sur-Ivette near Paris, who worked also in genetic recombination in bacteria (I will not say the name that I remember perfectly), I was asked what a lysosome was (but I remember also a similar surprising question from the eukaryotic part when a good biochemist, I remember also the name, asked me once, while showing him an electron microscopy picture of yeasts, if they really had nuclei!). However, I liked my work and I am happy that I did not change.

DJK: I must interrupt for a moment because this reminds me of a comment from Ana Maria Cuervo (Albert Einstein College of Medicine), about the time she interviewed with you regarding the possibility of obtaining her PhD under your supervision: “I am still amazed (and tremendously thankful) that Erwin took me in his lab … he asked me ‘do you even know what a lysosome is?’ I could only say ‘umh … umh … ’ while trying to quickly get straight in my mind the difference between a lysosome and a liposome. Needless to say, Erwin stormed out of the room in that very unique charming way that he always made everything a ‘big drama’ and came back with the book The Cell by Alberts (the thickest book I had ever seen) and told me ‘come back once you have read it’. I still don’t know if that was his ‘master plan’ to get rid of me or to test how much I was willing to take challenges.” This fits with the view that I think most people had about the yeast vacuole prior to the development of the autophagy field – if they thought about this organelle at all, the view was that it was the cellular garbage disposal, and not very important. As Ana Maria Cuervo noted, she joined your group after the departure of the people who had been working on lysosomes: “The fellows that preceded me in the lysosomal project had left the lab before I joined, and I was left as the only ‘lysosome’ person in the group. One of the sentences I probably heard more from Erwin to me was ‘Ana Maria your thesis is dead, nobody cares about lysosomes anymore’.” Needless to say, you were not done working with lysosomes, but please continue with your story.

EK: The first international meeting I attended was a Symposium on Processing and Turnover of Proteins and Organelles in the Cell during the 12^th FEBS Meeting on July 1978 in Dresden, East Germany. My boss was invited to give a talk, but he was unable to come and sent me in his place (a never-ending story with him). For me, it ended being a nightmare, because of several reasons: i) this was my first international talk, the audience was quite large and the other speakers included Christian de Duve, who was the chairman of the session, Peter J. Garlick, Helmut Holzer, Alfred L. Goldberg, Peter Bohley, Samuel M. Rapoport (who was the organizer), Valentin N. Luzikov and Gottfried Schatz; ii) I lost my baggage between Valencia and West Berlin (fortunately I had the slides in a handbag with me), and was unable to recover it until I was back in West Berlin; and iii) I arrived, tired, at Dresden slightly before 4.00 a.m. and I had to walk to the far University residence a long way in the night. Prof. Rapoport, the Symposium´s organizer, was not happy when I communicated him the change in speaker, but he was kind enough to allow me to give the programmed talk on Mitochondrial Protein Degradation (I have very good memories of this old scientist, with whom I talked on various occasions and who, years later, sent to me the complete first part of Göthe´s Faust in a large and really nice East German edition with beautiful illustrations. The book included a dedication from him and his charming wife, the pediatrician Ingeborg Rapoport, who told me that she was the first German girl born in Cameroon while it was a German colony, this means before the first world war, 1914–1918). In my presentation I was quite excited and enthusiastic, but, as far as I remember, the audience was not especially impressed with what I presented, probably because the mitochondria field headlines were then mitochondrial transport of proteins and non-lysosomal degradation mechanisms of mitochondria in reticulocytes and other cells. Anyway, in spite of my fears (for example, the night before my talk I was quite nervous since I was alone in the dormitory and I did not know anyone there, so I was fully unable to sleep, which did not contribute to improve my general aspect, by showing up, in this quite formal environment, typical in East Germany, still in my travel attire), it was a very good experience for me. However, and although I realize that attending scientific meetings is probably very important in a scientist’s career, since then I don’t like it very much. I especially hate the long waits at various airports when flying from Valencia (connections have only recently improved) and I have tried to reduce this important part of the scientific activities to a reasonable limit.

DJK: Apparently, your feelings about traveling are well known. Patricia Boyá (Consejo Superior de Investigaciones Científicas) commented, “He complains a lot … about being invited to conferences and then once there he enjoyed a lot being with people.” According to Patrice Codogno (Institut Necker-Enfants Malades), “Erwin’s particularity is his reluctance to travel. I was told early of this feature by Ana Maria Cuervo and Patricia Boyá. I experienced it directly on the way back from a GRC organized in Ventura in 2008. We were early at the LAX airport with Erwin, Patricia Boyá and Fred Meijer to catch our planes. With Fred and Patricia, we were seated and chatting in a relaxed manner. Erwin was standing in front of us nervously pacing the floor just like Robert De Niro in the Martin Scorsese movie ‘Raging Bull’ before stepping into the ring for a boxing fight.”

EK: After the meeting in East Germany, I continued my work on the lysosomal role in mitochondrial protein degradation. One example of these studies involved using immunogold procedures at the electron microscopy level with specific antibodies, which we had to prepare ourselves from New Zealand white rabbits, because in those times very few commercial antibodies were available. l noticed for the enzymes carbamoyl phosphate synthetase I (CPS I), ornithine transcarbamoylase (OTC) and glutamate dehydrogenase (GDH) that the mitochondria enclosed in the autophagosomes contained approximately the same number of gold particles per square micron of mitochondrial area than those that remained free in the cytoplasm, suggesting that autophagic vacuoles degrade mitochondria with their full protein content. Since once a mitochondrion is inside an autophagic vacuole it is quickly degraded with a half-life of about 8 min, as first shown, I think, in 1978 by Ullrich Pfeifer, it appeared that all mitochondrial proteins within an autophagic vacuole should be degraded at approximately the same rate. However, the half-lives of mitochondrial proteins were found to be quite different, ranging from a few minutes to various days and this was a strong argument frequently employed then against the importance of autophagy in mitochondrial protein degradation. Although the differences in mitochondrial protein half-lives could be due in some cases to differences among the experimental procedures used to measure this parameter, the group of Prof. Santiago Grisolía (Figure 3) measured in the laboratory with the same procedure the half-lives of the three above-mentioned proteins in rat liver and found that while GDH had a half day of about 1 day, CPS and OTC had half-lives of about 8 times longer. Therefore, and although, using the electron microscope, mitochondria were frequently found inside autophagic vacuoles, the vast differences in these and in other half-lives of mitochondrial proteins appeared to be inconsistent with autophagy of whole mitochondria as the main mechanism for their degradation.

Figure 3.

Erwin Knecht, a visitor from Kansas City University, and Prof. Santiago Grisolía, circa 1986. Photo courtesy of Asunción Montaner.

As an alternative, there were mitochondrial proteases, but some of them were either found to be contaminants from other organelles present in the isolated mitochondrial fraction, or in those cases that they were genuine mitochondrial proteinases, only a low or a limited proteolysis was apparently shown. Therefore, we reasoned that a possibility that could solve this incompatibility, namely “in bulk” autophagic degradation of mitochondria and different half-lives of their proteins, was the existence of mitochondrial heterogeneity. Thus, if populations of mitochondria existed in tissues with different proportions in their content of specific proteins and those mitochondria containing higher amounts of enzymes with short half-lives were preferentially degraded by autophagy (I considered the possible existence of a specific autophagy, well aware that this was against the general belief that autophagy was nonselective and that lysosomes did not discriminate what was being degraded), this could explain the differences in the half-lives of mitochondrial proteins. Therefore, we investigated this possibility in rat liver for the three enzymes whose half-lives had been measured with the same procedure, it est one with a half-life of 1 day and two with a half-life both of about 8 days. We first carried out double immunogold experiments in which we simultaneously labeled two different mitochondrial enzymes, one with a large gold particle and the second with a small one and vice versa. We found that the proportion of molecules, estimated by counting the number of gold particles, of CPS I vs GDH, of OTC vs GDH and CPS I vs OTC was quite similar in the different mitochondrial sections of a same cell. The counts revealed an approximately constant relative proportion of these proteins within a single hepatocyte, i.e. homogeneity among mitochondria and, thus, no clear possibilities to investigate a specific autophagy of certain mitochondria. Therefore, these observations did not solve the question of why the half-lives of mitochondrial proteins are so different in rat liver if autophagy has a prominent role in their degradation.

However, using immunochemical procedures in the rat liver tissue, it was found that the levels of mitochondrial enzymes vary in different zones of the liver acinus. With our three enzymes, it was found that, for example, CPS I and also OTC were present at higher levels in periportal areas, while the opposite occurred with GDH, since its levels were lower in these areas and higher in others. Thus, with the help of my first PhD student, Jose Luis Vargas, who unfortunately died after obtaining his PhD in a car accident, we isolated hepatocytes and, based on previous reports from other laboratories on the separation of hepatocytes from different acinar zones, we obtained in a Percoll gradient various population of cells that we analyzed by flow cytometry and determined in each the content in the three enzymes by western blot, and the autophagic activity by pulse-chase experiments with inhibitors of autophagy. We found in light hepatocytes higher levels of GDH, which has the shortest half-life of the three mitochondrial proteins we analyzed, and more lysosomal activity, while this activity was comparatively lower in heavier hepatocytes with higher levels of CPS I and of OTC, both with longer half-lives. Also, we isolated mitochondria and lysosomes/autophagic vacuoles from rat liver after lysosomal inhibition. Then, we analyzed by immunoblot the levels of these proteins. We found that the relative content of CPS I and of OTC in the isolated autophagic vacuoles was lower than the content of GDH, which was about three times higher. Therefore, we concluded that it is possible that the differences in the levels of mitochondrial enzymes and in autophagic activity in different acinar zones of the liver could contribute to explain, at least in part, the differences in half-lives of mitochondrial proteins in rat liver.

DJK: It is quite interesting that you mention the different turnover rates of mitochondrial proteins, based on studies you were carrying out in the 1980s. The mechanism that allows such a difference has remained a topic of interest. For example, Hagai Abeliovich and Joern Dengjel examined the segregation of mitochondrial matrix proteins in an article published in 2013 [5], approximately three decades after your own work.

EK: Once we found an apparent mitochondrial homogeneity in the composition of three mitochondrial enzymes in the same cell, but hepatocyte heterogeneity in the levels of these enzymes and in the lysosomal degradation activity along the liver acinus, our curiosity was satisfied and we were not interested to repeat this with other enzymes, which was the obvious continuation of the work. Therefore, we changed to another topic, leaving these results to be further developed by others or to be simply rejected. Segregation of mitochondrial proteins at certain mitochondrial sites for selective degradation by mitophagy in yeast, as proposed by two reliable workers like Abeliovich and Dengjel, is certainly an interesting possibility, but we were unable to notice this in the mitochondrial ultrathin sections of single hepatocytes with the three investigated enzymes and using double-immunogold labeling in electron microscopy, which I think was the most appropriate of the available procedures at that time to investigate the question. I don’t know in yeast cultures in which the different cells should be more similar, but anyway I think that Abeliovich and Dengjel’s proposal is an interesting possibility to test in more detail.

DJK: Do you have a general philosophy that drives the way you view research?

EK: Well, as with most scientists I have always considered that research is a fun job with a wide range of different and interesting questions and, therefore, once I started a problem I was immediately interested in other aspects, jumping from a general question to another, probably without dealing in depth with any single one. Of course, this was a problem when writing a grant application, because I have always found it quite difficult to execute a scientific project of a grant application that has been written for a duration of let’s say three years and about one year before you start it, obviously if it was approved by the specific referees and the final evaluation committee. Then, if you don’t carry out all the experiments proposed in your project and get more interested in new aspects, you have to convince the evaluation committee of the reasons for not doing something and doing instead a different work and this is not always easy if your choice was based in simply being curious about that. This is only to mention the problems I have found many times, probably because my inconsistency to follow in depth a specific area when it appears a little boring to me and when I get interested in something different, and is not at all a criticism of the Spanish evaluation system. I have worked in the evaluation of grants from different countries (Canada, USA, UK, Germany, France, Argentina, etc.) and I recognize that the Spanish evaluation procedure of grants, fellowships and contracts, in which I have also collaborated in various Committees in Madrid for a long time, is quite fair and very well organized, except for being a little too strict in the follow-up of the original project. Anyway, my work has been always well supported (of course in terms of Spanish standards) with grants.

DJK: I have to say that I completely agree with what you have expressed here, and I am confident that many scientists have the same opinion. That is, it is often difficult to accurately predict where a project is going, and certainly hard to say where your scientific interests will take you. The flexibility to explore is very important with regard to making novel discoveries, but it is unfortunately somewhat counter to the expectations of the granting agencies or the review panels. Writing in a grant “Trust me, I will figure out what to do when I get there” is not likely to be well received. What do you consider your most important contributions to autophagy research?

EK: In my work I liked, and I have considered especially important for obtaining interesting results, to discuss with my students on the scientific problem and the best methodology to answer a specific question, and at the same time to try to help them to be good researchers in biology. Also, I learned to appreciate collaborations with other researchers, contributing, I hope, to their work with my own experience in protein degradation. Both aspects are what I most appreciate, and I think that they probably give the main value to my work in autophagy.

Concerning specific experiments, and although I am realistic enough to consider that our contributions to the field have been modest compared to others, what I consider our most satisfactory work, which may or may not be generally appreciated, are probably the following:

1) Specific lysosomal transport of proteins through its membrane for degradation.

2) Cross-talk of autophagy, proteasomes and other lysosomal and non-lysosomal mechanisms in the general degradation of short and long-lived proteins in cultured cells under different conditions,

3) Role of autophagy in some rare diseases.

DJK: Can you provide some specific details of these areas? For example, I know that you have had a long-time interest in direct transport of proteins into the lysosome; how did this fit in with the work from Fred “Paulo” Dice, which was being done at around the same period of time?

EK: I will start with my comments on the specific lysosomal transport of proteins through its membrane for degradation. When I realized that “in bulk” autophagy could probably not explain alone the different half-lives of intracellular proteins, I started around 1990 and simultaneously, since I had then two/three students in the lab, two areas of research for an explanation. One was obviously the possibility of a non-lysosomal mechanism, especially because of the works of Hershko, Ciechanover and colleagues on ubiquitin (see below point 2), but the other was the possible existence, in addition to the classical autophagy, of a specific autophagic degradation of proteins.

When I started to investigate this last possibility, I was aware of the work of Gottfried Schatz and of others, who studied the import of cytosolic synthesized proteins into mitochondria using an in vitro system with isolated mitochondria. In those times I had only pre-docs in my recently formed independent group, because post-doctoral fellowships were then here in Spain very scarce, and the majority of post-docs went abroad and/or to large labs. However, most of my pre-docs were hard workers and also very bright and I had also a quite efficient, but an apparently invisible (always working behind-the-scene), technician, Asunción Montaner, who ended up working with me for more than forty years, both before and after I could form my own research group, since I have always worked quite independently. Asunción facilitated very much my work, resolving quite efficiently many problems, not only technical but also bureaucratic (while she was working at the IIC, she finished her studies as a lawyer, but she continued working as technician), which is one of the aspects of this work that I, like many others, most hate. Therefore, I experienced no big problems without post-docs.

DJK: Allow me a brief interruption here – your technician studied to be a lawyer, but ended up remaining in your lab? I would not have thought the same type of interests would lead someone into these two different fields.

EK: I think, and probably she too, that any knowledge can be useful for different types of work. Anyway, and continuing the story, we tried to do the same as was done by Gottfried Schatz in Switzerland with mitochondria, using, however, lysosomes and specific cytosolic proteins instead of mitochondria and mitochondrial pre-proteins and taking into consideration that if the proteins entered the lysosomes they would be immediately degraded.

To obtain a reasonably pure mitochondrial fraction is relatively easy, but with lysosomes, due to the heterogeneity in size, weight and density, it is another story. So, we investigated first various procedures from the literature to obtain purified lysosomal fractions from rat liver and cultured cells. In the classical differential centrifugation, lysosomes are mainly found at the mitochondrial fraction and since mitochondria are much more abundant than lysosomes, they were found in these fractions only in small amounts. These amounts increased when de Duve’s light mitochondrial fraction was prepared at various speeds. Therefore, in all procedures to purify lysosomes we obtained, first, a light mitochondrial fraction by differential centrifugation, after investigating various increasing speeds to change the proportion of lysosomes to mitochondria, and to enrich the fraction in medium-sized lysosomes as much as possible (this was one of the studies that I already presented in Dresden years ago, as shown in the Proceedings book and that we continued then). This fraction was subjected to various gradients to separate different subfractions containing mitochondria or lysosomes. The isolated lysosomal fractions that we obtained were evaluated by electron microscopy and measuring marker enzymes and lysosomal latency. We tried different media and procedures, and the best results by far were obtained when we used one of these slightly modified from the original one from Wattiaux et al. [6] with metrizamide as the gradient medium (an expensive product in Spain that we obtained from a local representative of a Norwegian Company). We finally obtained lysosomal fractions with lysosomal enzymes purified 30–70 times, 3–5% yield and more than 95% latency (i.e., less than 5% broken). These fractions were used to develop the in vitro assay to investigate the transport of cytosolic proteins into lysosomes.

We incubated for short times either in vitro synthesized proteins (from mRNA obtained from their cDNA) in a reticulocyte assay or the purified proteins in an isotonic medium with the freshly isolated rat liver lysosomes at physiological temperature and pH. In this in vitro assay different protease inhibitors were used to avoid the degradation of the protein that had entered into the lysosomes and, after washing and re-isolating the lysosomes, an exogenously added protease was employed to eliminate the protein that was simply bound to the external face of the lysosomal membrane, leaving the protein taken up by the lysosomes intact. Finally, a detergent to destroy the lysosomal membranes served as an additional control to test the efficiency of the exogenous proteinase treatment. This in vitro system allowed us to distinguish, in the presence of a lysosomal inhibitor, the protein that had been taken up by lysosomes (exogenous proteinase-insensitive, unless the detergent was added), from that simply bound to the external face of the lysosomal membrane (exogenous proteinase-sensitive, with or without detergent). In addition, the protein associated with the lysosomes without the exogenous proteinase and detergent treatments corresponded, in the presence of the lysosomal inhibitor, to the protein that was inside plus that at the external face of the membrane of lysosomes. In the absence of the lysosomal inhibitor, the observed protein corresponded only to that bound at the surface of the lysosomal membrane. Therefore, the difference from the other represented the protein inside the lysosomes, which should correspond approximately to the amount previously shown in the lysosomes treated with the external proteinase in the presence of the lysosomal inhibitor. Perhaps it sounds a little bit complicated because of the different controls, but it worked fine when being careful and fast enough and if the lysosomal preparations were fresh and their latency better than 95%. The proteins were identified (and quantified densitometrically) in SDS-PAGE gels in two ways: with ³⁵S-methionine and fluorography with the in vitro synthesized proteins in the reticulocyte system or with specific antibodies and western blot with the purified proteins. Using this system, we found that the enzyme glyceraldehyde-3-phosphate dehydrogenase was apparently transported in vitro into the lysosomes through its membrane. We also tested other proteins including other glycolytic enzymes for which, however, we had less effective tools, except for phosphoglyceromutase, and concluded that specific cytosolic proteins could apparently pass through the lysosomal membrane with various import efficiencies to be degraded.

As you know, Paulo Dice choose for his studies on direct lysosomal transport ribonuclease A. Incidentally, I have to say that he was really a quite nice guy (we smoked together after the meetings’ sessions: he, and Patrice Codogno later, were usually my smoking partners) and I was very sorry to hear about his death. We chose initially this enzyme, glyceraldehyde-3-phosphate dehydrogenase, because, unlike ribonuclease A, it was a cytosolic protein, we had its cDNA, prepared with the help of the group of Maria Eugenia Armengod from my Institute, and we also had very good antibodies, both polyclonal prepared by us and monoclonal, prepared with the help of another group of the Institute led by Amelia Martínez-Ramón. All this was a long work that lasted various years and was started initially by two quite efficient pre-docs, Fernando Aniento and Enrique Roche, and later quickly followed by another pre-doc, Ana María Cuervo. Ana María was really brilliant in research, she was my first student coming from the Medical School in Valencia and she participated very enthusiastically in the work, even completely alone when the two other students read their PhDs and left the lab. The results were finally published in 1993 in JBC [7]. But shortly before the publication I presented some of the results, in 1992, in a poster at the 9^th ICOP Meeting in Williamsburg (VI), where my English friend, Jennifer Rivett (see later), also presented, in a talk, her own and our joint results on proteasomes. In Williamsburg I met for the first time Paulo Dice, who came to the poster and discussed our results in the light of his own studies. We decided to join efforts to compare both systems. To this end, Ana María Cuervo in my lab and members of the group of Paulo Dice at Tufts in Boston (I remember Stan Terlecky) worked in close cooperation in their respective laboratories, with short stages of Ana María at Tufts in Boston during the summer holidays. It was found, for example, using our in vitro system of lysosomal transport, that: A) ribonuclease A was also incorporated into isolated rat liver lysosomes and showed competition with glyceraldehyde 3-phosphate dehydrogenase in the transport; B) the lysosomes that were active in this transport were those that contained intralysosomal hsc73, which could be separated from the others in the metrizamide gradient; and C) lysosomes isolated from fed rats or from rats subjected to various periods of starvation (up to 88 h) were active in the specific transport of proteins through the lysosomal membrane, but this activity increased progressively with the starvation time (confirming here previous results published by the group of Paulo Dice).

Then, with some of the papers published, Ana Maria presented her PhD at the Medical School in Valencia and soon she moved, as a post-doc, to Paulo’s lab in Boston. Here I have to say that she is very, very bright, but in addition she is such an exceptionally hard worker that neither me, which I think was not exactly a lazy worker, nor any of the students I know and that really worked very hard have been able to work so many hours per week as she did.

DJK: I think it is fair to say that Ana Maria valued that time immensely. For example, she commented that being the only one working on the lysosome project, “ … ended up being such a good thing, because it was Erwin himself who had to train me on all the techniques on lysosomal isolation, transport, analysis. Doing experiments side-by-side with Erwin were the most stressful and at the same time fun and exciting memories of my PhD. I loved every second! I loved the “German” Erwin that had to inspect (one million times) that I had correctly prepared every single tube and instrument that we were going to need and that asked me to repeat what we were going to do and how in every second. But I probably loved even more the “Spanish” Erwin capable to improvise in the middle of the experiment when (often my fault) things were not going as planned to make sure we could still save part of the experiment and get usable conclusions. The “drama” around us doing experiments together was endless: we started at 5:30 am (so to not be bothered by others), instead of the main lab we went to a small one in the basement (because it was quiet and closer to the centrifuges), nobody was allowed to interrupt and if anybody dared to say “good morning” Erwin will scream “you are going to make the ‘little girl’ (Erwin’s nickname for me during my whole PhD) mess up the experiment and I will have to kick her out of the lab”. I know that from outside this looks like madness, but I could not think of a more efficient way to imprint in a very inexperienced grad student as I was the importance of planning, focus and ability to improvise when doing experiments. Of course, all those many hours pipetting side by side and waiting for incubations, were filled by the most interesting conversations with Erwin about every topic in life but of course a lot about lysosomes. What a luxury for a graduate student to be able to receive first-hand training and knowledge from somebody like Erwin!

EK: Almost immediately after she left, I was interested in other questions and, more important, my Institute started to depend on a Foundation from the local Health Ministry, so that we had to progressively consider to give priority, instead of to basic research, to health-related studies, which were considered more appropriate for the new Institution. Therefore, and as far as I remember, in addition to being invited by Ana María to be coauthor of one of her papers, because she incorporated in it a few results that she did in my lab on oxidized proteins, I published, after her departure and concerning this area of direct lysosomal transport of proteins, a pair of short papers with my former students Fernando Aniento and Enrique Roche mainly done by them in their labs and a really nice (at least to me) paper. In it, we took advantage once more of the work of Gottfried Schatz in mitochondrial transport. My first post-doc, Carmen Aguado, and a pre-doc, Natalia Salvador, used our in vitro system with lysosomes to analyze the effects on the lysosomal transport of the protein dihydrofolate reductase of the drug methotrexate, which binds with high affinity to the protein and stabilizes it in a folded conformation. For this work, we obtained also the advice on some technical aspects and also some reactives from a German scientist, Martin Horst, who by chance had worked before with Schatz. I met Martin during a talk that I gave at Göttingen University invited by Kurt von Figura (I also met there, for the first time, Paul Saftig, with whom I collaborated much later, so it was a quite productive talk for me). The protein was taken up by lysosomes without methotrexate, but not in its presence, suggesting that it entered into the lysosome only in an unfolded conformation. The paper was published in 2000 [8] and the lysosomal transport was called there, for the first time in one of our papers on lysosomal transport, chaperone-mediated autophagy. This term was first proposed and used, also in 2000 [9], by Paulo Dice, and I think either he or Ana Maria told me about this new name. Since we found it quite appropriate, we also incorporated it into our paper.

What I learned during these studies, for example, the isolation of relatively pure lysosomal fractions, has been also useful to start to investigate other problems. One of my last works in autophagy was related with this. Briefly, I have always thought that when you don't have new bright ideas, you can always do, as many scientists do, an “omics” approach that does not require, at least initially, to think much. Because of our prolonged experience in purifying lysosomal fractions for the chaperone-mediated autophagy studies, we tried to identify new proteins on the lysosomal membrane that regulate the lysosomal activity, reasoning that the signaling cascades that regulate lysosomal activity should eventually arrive to proteins on the lysosomal membrane. Therefore, we investigated the effects of two well-established regulators of lysosomal activity, insulin and/or amino acids, on lysosomal membrane proteins. Once the cells were incubated, without or with amino acids and/or insulin, two post-docs, Carmen Aguado and Maribel Sánchez-Piris, obtained the corresponding lysosomal fractions, prepared the lysosomal membranes, subjected their proteins to 2D-gel electrophoresis and identified the differences using a DIGE approach by simultaneously labeling the four samples with different fluorophores. The 2D-gels were analyzed with the DeCyder program and we found differences in the levels of various proteins in the lysosomal membranes from cells incubated with or without insulin and/or amino acids. For example, concerning the effects of starvation, we organized these lysosomal membrane proteins into three main groups: A) Subunits A, B, C and E of the vacuolar-type H⁺-ATPase, whose levels increased; B) three proteins related with Ca²⁺ signaling: annexin A1, and A5 and copine 1, whose levels also increased; and C) others, including two proteins related with the cytoskeleton, one protein related with protein synthesis (elongation factor 1 alpha, which I think the group of Ana María Cuervo identified also independently in another study) and two mitochondrial proteins, whose levels decreased, with the exception of one of the proteins related with the cytoskeleton that did the opposite. These were some of my last basic research experiments. Much later, Ghita Ghislat (one of the last members of my group, which in the last ten years included, at different times: four post-doctoral fellows, Carmen Aguado, Eva Pérez Jiménez, Jaime Cárcel and Juan Miguel Esteve; and six pre-docs: José Manuel Vidal, Alihamze Fathinajafabadi, Félix Moruno, Marcos Lahuerta, Ghita Ghislat and Rajaa Errafiy, the first and the next two were co-directed with me for their PhDs by Carmen and Eva, respectively, and the fourth by both of them). Ghita confirmed and investigated one of these groups and found that annexin A5, but also annexin A1 and copine 1 (results with these two are unpublished, because it was exactly the same as with annexin A5), whose levels on the lysosomal membrane decreased in the presence of amino acids and insulin, facilitated under starvation the fusion of lysosomes with autophagosomes but not with endosomes. If we had continued this work, we had also investigated, apart from the lipid composition of autophagosomes vs endosomes: i) the kinases that phosphorylate one of the ATPase subunits, because we found that this specific subunit and apparently not the others was phosphorylated in response to amino acids and I had acquired a reasonable knowledge of ionic pumps during my stay in Kalman’s lab, and ii) the role of elongation factor 1 alpha, even knowing that it would be quite difficult to compete with Ana María, since I think that an interesting problem is still to identify other signals that simultaneously control, in opposite directions, protein synthesis and degradation.

DJK: So, you and Paulo Dice ended up working on the same topic, which ultimately came to be called chaperone-mediated autophagy, completely independently? I guess that is similar to various labs that ended up working on macroautophagy, but initially studied the degradation of particular proteins (or in my case, the biosynthesis of a protein). What about the other two topics that you mentioned?

EK: Regarding the first part of your first question, yes, we worked independently on the same topic, but no doubt that the credit of the finally called chaperone-mediated autophagy mechanism belongs to Paulo’s and Ana Maria’s labs, since they published by far many more papers on the topic and described many more molecular details of the mechanism than my laboratory did. In fact, at a meeting in Turku, after my talk on the specific transport of cytoslic proteins through the lysosomal membrane, I was asked by an old researcher if I was a post-doc of Paulo. Since Paulo was less than two years older than me, I simply answered: Unfortunately for me, no.

Next, I will talk a little about our work on the cross-talk of autophagy, proteasomes and other lysosomal and non-lysosomal mechanisms in the general degradation of short and long-lived proteins in cultured cells under different conditions (Figure 4). The other possibility that I considered to explain the differences in the half-lives of proteins concerned a cytosolic protease, from which I learned more when I gave a talk in Earl Stadtman’s NIH laboratory while I was briefly in Washington for a FASEB Meeting. During a quite large and numerously attended party in 1989 with a Spanish colleague, Javier Cervera, at the Stadtman’s (Earl and Terry, both well-known biochemists, and the latter, was so kind to offer me an individual guided tour through the large and beautiful garden with a building containing a large climatized swimming pool) house near Washington after I gave a seminar at Earl Stadtman's laboratory. At the party I met a British young colleague, Jennifer Rivett, who had been working at the NIH, like other researchers, on a large multicatalytic proteinase. She was returning back to England to the University of Leicester first, and later to the University of Bristol, and we started a collaboration, in which the work in Valencia was carried out by a pre-doc, Amparo Palmer. For example, in our first joint paper we found by immunogold procedures the multicatalytic proteinase, which was later called (I think it was Alfred Goldberg at Harvard who coined the word) the proteasome, not only in the cytosol but also in the nucleus, where certainly there are no lysosomes. We noticed also a significant concentration of the protease at the outside of the endoplasmic reticulum, probably related with the role attributed later to the proteasomes in the production of antigenic peptides for presentation by the MHC complex class I, but to my satisfaction, because of the previous work in mitochondrial protein degradation, never inside mitochondria.

Figure 4.

Lab meeting, circa 1988. Photo courtesy of Enrique Roche.

Ana María Cuervo also participated in a specific aspect of this collaboration, in which we measured the half-life of proteasomes in rat liver and showed, with various procedures, that proteasomes were mainly degraded by autophagy [10]. Once again, we found out that, even in those ubiquitin-proteasome times, the importance of lysosomes should not be neglected. The joint work with the group of Leicester on the multicatalytic proteinase was continued by Amparo and later by other pre-docs, in particular by Graciela Fuertes, Inmaculada Esteban and Adoración Villarroya, who also investigated the cross-talk among the various pathways of intracellular protein degradation. They worked hard and precisely quantified, using many concentrations of inhibitors and treatment times, which they carefully analyzed before, the relative importance of proteasomes, autophagy, lysosomal mechanisms different from autophagy and other non-lysosomal mechanisms in the degradation in human fibroblasts of short-lived and long-lived proteins under different conditions (exponentially growing, confluent, short starvation and prolonged starvation). It was, for example, interesting to find out that, contrary to a general belief, lysosomes are also responsible of about 40–60%, depending on conditions, of the degradation of short-lived proteins. These procedures were one year later (in 2004) employed, in a collaborative paper with Paul Saftig and Eeva-Liisa Eskelinen; the first has moved from Göttingen, where I met him for the first time, to the University of Kiel in North Germany, to investigate possible alterations in proteolytic functions in lamp1 lamp2 single- and double-deficient fibroblasts [11].

Finally, the role of autophagy in rare diseases. Although we were essentially a basic research laboratory in Cell Biology, as I have already said, at the end of the XX siècle our Institute was not supported anymore by the bank and started to depend entirely on a private Foundation of the Health Ministry of our Local Government. Then, it was wise to re-direct progressively our basic research toward more translational aspects of biomedicine. Therefore, we started working with diseases, first with quite prevalent ones, like cancer or familial hypercholesterolemia. In this latter case, with a post-doc, José Javier Martin de Llano, and a pre-doc, Enrique Andreu, and with the collaboration of Miguel de la Guardia from the University of Valencia, we developed a new and very nice non-radioactive and sensitive procedure to investigate binding, transport and degradation of the LDL receptor. This procedure was useful both to identify LDL receptor defects as well as to quantify endocytosis. We used LDL-gold particles that were quantified, initially with inductively coupled plasma-mass spectrometry and, later, to allow the use with more accessible equipment of the hospitals, with electrothermal absorption spectrometry. All this work was published [12–14], but before, in 1997, I participated as a speaker at the International Workshop on familial hypercholesterolemia and the LDL-receptor in Amsterdam, to present the new procedure. However, I don’t know if it has been employed in some hospital with familial hypercholesterolemia patients.

Much later, in 2007, our laboratory was selected with other Spanish laboratories to be a member of a public Spanish virtual consortium called CIBERER, or Center for Biomedical Network Research on Rare Diseases, by its director Dr. Francesc Palau. This Institution has paid since then the salary of one of the members of my lab, Carmen Aguado, and, for a few years, also that of a technician, Nuria Mas, who worked with her. Within the CIBERER, we have investigated the relevance in their pathogenesis of possible alterations in the main pathways of intracellular protein degradation, in other words proteasomes and autophagy. Specifically, we worked in neuronal ceroid lipofuscinoses, retinitis pigmentosa, Lafora disease, X-adrenoleukodistrophy and mitochondrial diseases. The reasons why we choose those among the approximately 8,000 different rare diseases known in  those times, of which only a few hundreds had any treatment, was that we tried to apply our basic knowledge on the pathways of intracellular degradation to those rare diseases in most of which non-degraded material accumulates, because this was an indication of a possible defect in the biogenesis and/or in the degradation mechanisms. This accumulated material includes proteins in neuronal ceroid lipofuscinoses, polysaccharides in Lafora disease, lipids in X-adrenoleukodistrophy, and altered mitochondria in mitochondrial diseases.

DJK: One of the other investigators who participated in CIBERER, Pascual Sanz (Instituto de Biomedicina de Valencia) expressed, “In my opinion, Erwin has been the leader and referent in Spain on the autophagy field and in his lab, great scientists of the area had their initial steps … [Our] collaboration was supported by the participation of our labs in the Spanish network of excellence in rare diseases, named CIBERER. Different scientists have benefited from the participation of Erwin in this network since he was always willing to participate in collaborative actions between the members.”

EK: Since the CIBERER encouraged collaborative science, we had the opportunity to work with many excellent basic and clinical groups. I will comment on only two examples of that work with rare diseases.

The first refers to a pathology called Lafora disease for the Spanish doctor who described it in 1911, a fatal autosomal and recessive neurodegenerative disorder mostly caused by mutations in either of two genes: EPM2A, encoding laforin, a dual-specificity phosphatase with a carbohydrate binding domain, and EPM2B, encoding malin, an E3 ubiquitin ligase. The disease is characterized by the accumulation of polyglucosan, a poorly-branched form of glycogen, forming Lafora bodies in the cytoplasm of neurons and of other cells in peripheral tissues. Although it was proposed that Lafora disease could be an error of carbohydrate metabolism, we considered the possibility that a deficiency in autophagy may be also a disease feature that contributes to the accumulation of polyglucosans in Lafora bodies. While Carmen Aguado in my lab, in collaboration with the groups of Santiago Rodríguez de Córdoba in Madrid and of Pascual Sanz in Valencia, carried out these experiments, we learned from my colleague Patricia Boyá in Madrid that Sovan Sarkar, from the group of David C. Rubinsztein, was also interested in this question. Thus, I contacted Prof. Rubinsztein and we started a short collaboration, which was a real pleasure and quite rewarding, at least for me, because I found Dr. Rubinsztein to be a nice and quite helpful and understanding person, and a superb scientist. As a result, we obtained data in epm2a knockout mice and in cell culture systems showing that laforin is a positive regulator of autophagy and suggesting, therefore, that a defect in autophagy may contribute to Lafora disease [15,16]. Unfortunately, the applied work carried out later with activators of autophagy in mouse models of the disease to find a possible treatment for it has been unsuccessful. Our lab continued the collaboration on other aspects of Lafora disease with the initial Spanish CIBERER groups until my retirement, and we published various papers also with them and other CIBERER groups, in particular with those of Roser González Duarte, professor of Genetics at the University of Barcelona and an expert in retinitis pigmentosa, and Federico Pallardó, at that time dean from the Medical School of the University of Valencia, and an expert in oxidative stress.

DJK: You mentioned various collaborations, and I would like to interrupt for a moment, simply to note comments from some of your past collaborators. These all have a common theme, which anyone reading them can easily discern. For example, Roser González Duarte (University of Barcelona) wrote, “He is a high grade intellectual and great professional for his enormous ability to get to the bottom of topics and design new experiments to find the right answer.” Similarly, Santiago Rodriguez de Cordoba (Consejo Superior de Investigaciones Científicas) stated, “Erwin is very special: a superb scientist, very honest and reliable and at the same time extremely modest and quiet.” Paul Saftig had his own twist on your expertise, stating, “Erwin is still belonging to the ‘old style’ (meant in a positive way!) biochemists who are familiar with classical biochemical assays … I always appreciated his very careful and systematic way of performing experiments.” Yet another comment was from Carlos López-Otín (University of Oviedo): “I have always considered him as a superb scientist with a strong commitment for exploring the real value of experimental science.” Suffice it to say that you are held in very high regard by the many people who have worked with you over the years. Okay, back to disease connections.

EK: The second example refers to X-linked adrenoleukodystrophy (X-ALD), a severe and often lethal neurometabolic disease in which very-long-chain fatty acids (VLCFA) accumulate in the cytosol of the cells of patients. This is due to the loss of function of the peroxisomal membrane transporter ABCD1, which transports very-long-chain fatty acids to this organelle for degradation. There are variants of the disease, but in its more severe forms it produces progressive demyelination in the central nervous system, axonopathy in the spinal cord and adrenal insufficiency. The work was done in collaboration with the group of Dr. Aurora Pujol at the IDIBELL in Barcelona, which is a reference laboratory in the research and treatment of this pathology.

Since we knew from the work of other laboratories that neuronal autophagy is important for axonal homeostasis, we investigated if it was altered in X-ALD. Using different procedures (electron microscopy, GFP-LC3, western blot and pulse-chase experiments) with various models of the disease (affected and non-affected brain areas and fibroblasts subjected to different treatments from X-ALD patients and spinal cords of abcd1 null mice of different ages and controls thereof) we concluded that autophagy is impaired in these disease models. Next, we asked why this defect occurs and which was the signaling mechanism responsible for the observed autophagic impairment, and found that it was directly due to the accumulation of VLCFAs and the activation of MTORC1. Finally, we analyzed the effects of injecting intraperitoneally into X-ALD mice an inhibitor of MTORC1, the rapamycin ester temsirolimus. As expected, X-ALD mice treated with this inhibitor showed an inhibition of MTORC1 activity, and an activation of autophagy, but we also investigated the consequences of this treatment. Previous studies from Aurora demonstrated that oxidative stress contributes to the progression of X-ALD, resulting in an accumulation of oxidized proteins, a decrease of ATP levels and alterations in proteasomes, including increased chymotrypsin-like activity and increased expression of the immunoproteasome subunits LMP2 and LMP7 [17]. Next, she found that temsirolimus normalized all these changes. X-ALD mice have a neuropathological phenotype characterized by increased labeling with several markers of oxidative DNA damage, microgliosis, astrocytosis, axonal damage and the presence of scattered myelin debris. Treatment of the X-ALD mice with temsirolimus reduced the alterations of these markers in mice to wild-type levels and was also found to stop the progression of locomotor deficits associated with X-ALD mice. So, in summary, in X-ALD, an excess of VLCFA produces ROS and causes an accumulation of oxidized proteins and damaged organelles, as well as impairment in autophagic flux, via MTORC1 activation. All these and other alterations result in axonal degeneration. The MTORC1 inhibitor temsirolimus restored autophagic flux and, as a consequence, prevented the accumulation of oxidized proteins, the bioenergetic failure and other alterations, leading to protection against axonal degeneration and the associated locomotor disability in the X-ALD mouse model. Therefore, we proposed in a paper in 2015 [18] that this treatment is an attractive therapeutic option for X-ALD patients and, in fact, we got a patent for that and, based on our results, the European Medicine Agency approved temsirolimus in 2016 as an orphan drug for the treatment of X-ALD.

Finally, I want to refer to what I mentioned at the beginning of this point, namely my work with students and with other laboratories. I really don’t know if I was a good mentor in the lab or not. Anyway, I tried to do my best to bring out also the best from my students and I liked it. In fact, since 1998 I have combined the work at the lab and in the University, working as an associate teacher of Biochemistry at the public University and of Cell Biology at a private University, because I think that it is important that we, as researchers, try to transmit our enthusiasm for science to the young future researchers in the University. Concerning my students in the lab, I have followed their careers and certainly I know that some of them work now with success in various sites and in different problems of research, including protein degradation. I do not know if I was of some help for them, apart from facilitating them to get their PhDs, but I am completely sure that I never could have accomplished any result without the invaluable help of them and of my technicians, and for that I am very, very grateful to all of them.

DJK: I think the value imparted to the students under your supervision comes across in a statement from Ana Maria Cuervo, who commented “Training under Erwin looked very different ‘from the outside’ to what was really going on. From outside it may look like a hectic place with a lot of drama and students living ‘in fear’ of not being up to the challenge. From inside, it was all about science and our training and about creating the most long-lasting trust that should govern all scientific teams. Our aspiration to gain Erwin’s trust was the daily drive to want to do better and to keep going despite all the ups and downs in the project. The love for ‘the answer’ to the scientific question is the best gift that any mentor can pass to their students, and nobody has been better in doing that than Erwin, in his very unique and unconventional way and always with a ‘sparkle’ with his witty sense of humor.”

EK: I have also learned the pleasure of collaboration with other groups. I remember, in Spain, among the most recent collaborations outside my research center (sorry if I forgot to mention someone), Pascual Sanz, Federico Pallardó and José Ramón Penadés in Valencia, Santiago Rodríguez de Córdoba and Patricia Boyá in Madrid, Roser González-Duarte, Aurora Pujol and Gustavo Egea in Barcelona, María Eugenia Armengod in Valencia and Carlos López-Otín in Oviedo. In other countries I have to mention my collaborations with Paulo Dice in the USA, David Rubinsztein and Jennifer Rivett in England and Paul Saftig and Martin Horst in Germany. I hope that my collaboration helped in same way the work of these colleagues, and I am sorry if not. From my point of view, although the results of all these collaborations produced sometimes positive but sometimes also negative experimental results, in all cases the personal interaction and the input of new ideas from all of them was quite satisfactory to me. Also, I enjoyed always my conversations with other scientists at the autophagy and other meetings in which I learned new things and got new ideas. In my last meeting, I was convinced by Ana María Cuervo and Patricia Boyá (both know that I don´t like to attend meetings very much) to present a talk on my old research at a workshop on the “History of Autophagy” in Tübingen, held from the 14 to 16 December 2016. I suspect that I was invited to it because Ana María was unable to attend it, but anyway I liked very much this meeting that was my last one outside Spain. In Tübingen I had the opportunity to meet again various European colleagues, like Sharon Tooze (I know she is a nice American lady in England that can be considered also a European), Patricia Boyá, Eeva-Liisa Eskelinen, Michael Thumm, Ullrich Pfeifer, and especially Alfred Meijer and Patrice Codogno with who I had always a quite good relation since our first joint meetings. But at the same time, I met for the first time personally other new and quite nice colleagues that I never met before: especially Fulvio Reggiori and Tassula Proikas-Cezanne, who organized the meeting, and also Nicholas Ktistakis and Anne Simonsen. It was really a quite nice end for my attendance to international meetings and I thank very much the organizers for their invitation.

DJK: It should be quite obvious to anyone reading this article, and I am certain is clear to people who know you, that you were quite serious about research. One of your former PhD students, Félix Moruno (currently at the University of Texas Health Science Center), commented “His office was built into the lab, so he was aware of everything that happened in the lab … he liked research so much, and he preferred to invest as much time as he could in his lab, and not doing paperwork … even in his 60s, he liked to spend some time working on the bench with his Research Assistant” (Figure 5).

Figure 5.

Completing the dreaded but necessary paperwork in his office in Valencia, circa 1983. Photo courtesy of Asunción Montaner.

What do you personally find most interesting about the autophagy field today? Where do you see the field heading?

EK: After the initial conferences in East Germany on “Proteolysis and Protein Turnover”, which I usually attended, and the 11^th conference of 1996 in Turku (Finland), Prof. Ohsumi and his Japanese colleagues started, as you know, organizing a first international symposium on autophagy celebrated in Okazaki (Japan) in 1997. This symposium was continued in 2000 by a second symposium organized in Aix-les-Bains (France) by Patrice Codogno, followed by others in Osaka (Japan) in 2002 and in Mishima (Japan) in 2007, and since 2003, a series of Gordon Research Conferences on Autophagy in Stress, Development and Disease, first in Maine (USA), then in Il Ciocco (Italy) and later in other places (Ventura, CA in 2008 was the last one I personally attended). Since the meeting in Turku and in all the following meetings, I was always strongly impressed by the excellent work with yeast, from that I heard for the first time, of several laboratories in the USA (Daniel Klionsky), Japan (Yoshinori Ohsumi) and Europe (Michael Thumm) on the identification of the various proteins implicated in the different steps of autophagy and related processes. I clearly understood that compared to my own and similar works, this really represented a qualitative jump in autophagy that also opened new avenues for the research with mammalian cells. In fact, these studies anticipated the steady increase, since about 2005, of the number of laboratories working in autophagy and of the corresponding number of publications to reach the actual dimensions and the popularization of the word “autophagy”, which is the name of this journal. I never had expected in my first years of work something like that happening.

Of course, what I find most interesting today on autophagy is related with my own preferences (Figure 6) and my work with mammalian cells, and does not necessarily have to represent a well-accepted direction for where the field is heading. For example, I think that an interesting area of research is to continue with the identification of the various proteins, especially those that are transitory components of the autophagic machinery, to facilitate or inhibit some of its functions, and also of the lipids implicated in the various forms of autophagy. One of our last studies was in fact focused on that direction (see above). The specific regulation of all these components is quite appealing to me, not only as a basic research but also to develop pharmacological applications of interest in human health.

Figure 6.

Erwin Knecht, as he preferred to spend his time, circa 2017. Photo courtesy of Enrique Roche.

Closely related with these studies and also quite important in my modest opinion are those on the role of the various types of autophagy (macroautophagy, microautophagy, crinophagy, chaperone-mediated autophagy and probably others) in important human processes, especially aging and the different brain functions, as well as the relevance of all these processes in the thousands of human pathologies. I believe that some modulators of these autophagic processes, not only pharmacological but also natural, can be quite useful for the human health, at least in those cases where the role of autophagy is investigated in depth in specific human diseases.

Another area of promising (in my opinion) research refers to the inter-regulation and crosstalk for the degradation of intracellular proteins in particular and for proteostasis in general under various physiological and pathological conditions of the classical autophagy (macroautophagy) and other lysosomal degradative mechanisms (chaperone-mediated autophagy, microautophagy, etc.) and proteasomes and non-lysosomal and non-proteasomal protein degradation mechanisms in the cell (calpains, organellar proteases, etc.).

Finally, most work on the topic of autophagy refers to intracellular protein degradation. However, in contrast to proteasomes, which have only three proteolytic activities, lysosomes contain many other degradative enzymes potentially able to degrade lipids, polysaccharides, nucleic acids and almost any cell component. Therefore, I find it probably interesting to investigate in more detail the specific mechanisms of autophagic degradation of cell components different from proteins, as well as to know the role of this degradation in various functions and their relevance in specific pathologies. I would be particularly interested in the degradation of lipid droplets, but especially in a possible regulated microautophagic degradation of glycogen.

DJK: All of this is quite interesting; however, I am also hoping that readers of this interview can get some additional insight into the personal side of Erwin Knecht. Along these lines, Félix Moruno related the following: “He also has a funny side, hidden somewhere, which is not well understood by everyone. Erwin hates long meetings and presentations, and he does not hide his boredom. He could speak to you with his masculine voice and gradually make it lower and lower, whispering, until his comments were inaudible, only moving his mouth pretending he was still talking, making you feel that you were getting deaf. He enjoyed doing that just to see how we reacted.” Personally, I find this quite humorous, as it sounds like something I might do. At any rate, this comment was echoed by Roser González Duarte, who noted, “His harsh irony and enormous critical capacity inspired and enriched many conversations, and filled with humor the free time during the CIBERER meetings, where we frequently met.” Similarly, Guillermo Mariño (University of Oviedo) wrote, “Apart from certainly being a top scientist and being extremely committed to science, I would emphasize his sense of humor, which is an essential part of his character. As you surely know, he is a Spanish scientist with German roots, and this is a concept he often makes funny jokes about.” So, what would you care to tell us about your sense of humor?

EK: I realize now that I probably have been always too critical with almost everything and everyone in science and also outside it, including with myself and my own mistakes. But I was educated in that way and if you decide to work in science, which is a very enjoyable and funny election, you will find that it is also a quite difficult game to play, with so many problems and annoyances that sometimes, and to avoid to become a too bitter person, you have to use joking of the situations and irony as a defensive mechanism of your mental stability.

DJK: Clearly there is the “intimidating” side of Erwin Knecht, but also the supportive side. According to Patricia Boyá, “he is a very sincere person that speaks very directly, and he might give a hard impression, but on the inside has an amazing heart. He has helped me a lot in my scientific career, always giving frank advice and been very supportive.” Along these lines, Guillermo Mariño commented, “Erwin is really a person that once you know him you don’t forget easily. He is extremely committed to science and always cares for and supports other people when they are in not optimal situations.”

EK: Like many other scientists, I believe that science strongly relies on young people with original ideas. Therefore, and logically, if I met a brilliant young scientist coming back to Spain, I, like other colleagues, have tried to support, within my limited influences and experience, that, instead of being integrated into another lab, he or she could form their independent lab and get their own grants to develop new ideas and refresh a little bit the air. Also, with the same logic, I like to follow their careers and try to help them in some way if required. However, I don’t know and always doubt if my assistance and advice, which obviously could be wrong like that of others’, has been really of any help for them.

DJK: I am sure it has, Erwin.

Funding Statement

This work was supported by the National Institute of General Medical Sciences [GM131919].

Disclosure statement

No potential conflict of interest was reported by the author(s).