Ubiquilin at a crossroads in protein degradation pathways

Abstract

Ubiquilin proteins are conserved across all eukaryotes and function in the regulation of protein degradation. We found that ubiquilin functions to regulate macroautophagy and that the protein is also a substrate of chaperone-mediated autophagy.

Ubiquilin proteins are present in all eukaryotes and appear to function in protein degradation pathways. Humans contain four ubiquilin genes each encoding a separate protein. The proteins are approximately 600 amino acids in length and share extensive homology with one another. They are characterized by an N-terminal sequence that is very similar to ubiquitin, called the ubiquitin-like domain (UBL), followed by a longer, more variable central domain, and terminate with a conserved 50-amino-acid sequence called a ubiquitin-associated domain (UBA). This structural organization is characteristic of proteins that function to deliver ubiquitinated proteins to the proteasome for degradation. In accordance with this function, the UBL domain of ubiquilin binds subunits of the proteasome, and its UBA domain binds to polyubiquitin chains that are typically conjugated onto proteins that are marked for destruction. Indeed, we recently showed that ubiquilin is recruited to the endoplasmic reticulum where it binds and promotes the degradation of misfolded proteins to the proteasome during ER-associated degradation (ERAD).

Remarkably, ubiquilin was also recently reported to be involved in macroautophagy. The finding was based on colocalization of ubiquilin with autophagosomal marker LC3 in cells, and because overexpression of ubiquilin-1 suppresses and silencing of its expression enhances, starvation-induced cell death. In our recently published paper we describe our evidence linking ubiquilin to autophagy. We demonstrate that ubiquilin is indeed present in different structures associated with macroautophagy and that it is required for a critical step in autophagosome formation. Additionally, we also demonstrate that ubiquilin is a substrate of chaperone-mediated autophagy. The findings suggest that ubiquilin might play an important, and perhaps a crucial, role in dictating the pathway of protein degradation in cells.

In previous studies we found that ubiquilin proteins expressed in normal growing HeLa cells are very stable with a rate of turnover in excess of 20 h. Because most long-lived proteins are degraded by autophagy, we felt it was important to distinguish whether ubiquilin localization in autophagosomes was simply related to the expected route of degradation of the protein or whether it was related to some special function in autophagy. Accordingly, our experiments were designed to distinguish between these two possibilities.

Using double immunofluorescence microscopy we found that endogenous ubiquilin and LC3 proteins are present in puncta in HeLa cells. To ensure this was not an artifact of the staining procedure, we cotransfected HeLa cells with ubiquilin-1 and LC3 expression constructs that were tagged with either mRFP or GFP proteins and again found that the two expressed proteins are colocalized in puncta, irrespective of which tag was fused to the proteins. Further evidence supporting ubiquilin localization to autophagosomes was obtained by showing strong enrichment of ubiquilin proteins upon purification of autophagosomes from mouse liver and by the strong immunogold staining of the protein in autophagosomes in mouse brains in a transgenic mouse model of Alzheimer disease.

To determine if ubiquilin localization to autophagosomes is mediated by interaction with LC3 we conducted immunoprecipitation experiments to examine whether the two proteins coimmunoprecipitate with each other. Indeed, our results showed that the two proteins coimmunoprecipitate with one another, indicating that they bind together in a complex. However, we did not detect any strong binding between bacterially expressed forms of the proteins, suggesting that the interaction between the proteins in cells might be mediated by a bridging factor(s).

We next used a pH-sensitive tandem-tagged mCherry-GFP-LC3 reporter that is used to monitor maturation of autophagosomes to autolysosomes to determine whether ubiquilin is present during the different steps of macroautophagy. Indeed, we found that anti-ubiquilin staining is present throughout the different structures involved in the process, and interestingly, we also noted that the structures are enriched for K48- and K63-ubiquitin linkages. Because ubiquilin contains a UBA domain that binds ubiquitin chains we examined whether proteins containing K48- and K63-ubiquitin linkages coimmunoprecipitate with ubiquilin. Indeed, our immunoblots indicated that proteins containing both of these types of linkages coprecipitate with ubiquilin, consistent with the idea that ubiquilin might target proteins with diverse ubiquitin linkages for degradation by autophagy.

To determine if ubiquilin is required for autophagy, we knocked down the ubiquilin-1 and -2 proteins in HeLa cells (which mainly express these two ubiquilin isoforms) by siRNA transfection and examined if loss of the proteins altered LC3-I and LC3-II levels. Interestingly, we found that ubiquilin knockdown over a 72 h time period is associated with a progressive increase in LC3-I levels and a concomitant decrease in LC3-II levels. Furthermore, ubiquilin knockdown led to an ∼45% reduction in the number of cells containing five or more autophagosomes. Based on these results we propose that ubiquilin is required for maturation of LC3-I to LC3-II, which we speculate might be related to the requirement of the protein in macroautophagy.

We next asked if ubiquilin protein is consumed during autophagy. We examined this by treating HeLa cells with puromycin to induce protein misfolding and macroautophagy. Immunoblot analysis of the protein lysates examined at 2 h intervals over a 7 h period of exposure to puromycin revealed a direct correlation between stimulation of macroautophagy and a time-dependent decrease in the ubiquilin and LC3-II protein levels. The time-dependent decline in the proteins is inhibited by treatment of cells with two different autophagy inhibitors, 3-methyladenine and bafilomycin A₁. The results suggest that ubiquilin protein is consumed during macroautophagy.

The consumption of ubiquilin during macroautophagy prompted us to examine if ubiquilin might also be involved in chaperone-mediated autophagy (CMA), which involves the active transport of proteins into lysosomes. Support for this idea arose because ubiquilin proteins contain two sequences that conform to a pentapeptide motif involved in CMA. An in vitro CMA assay using recombinant GST-ubiquilin-1 fusion protein and purified lysosomes confirmed ubiquilin is an active CMA substrate. The results suggested that ubiquilin can be consumed by two different types of autophagy, macroautophagy and CMA. We speculate that this dual mode of consumption may provide a potential switch whereby changes in ubiquilin levels beyond a certain threshold might trigger execution of either macroautophagy or CMA. The idea that such a switch exists stems from previous work that showed inhibition of CMA can lead to activation of macroautophagy and vice versa.

Several intriguing new questions emerge from this and previous works, including what exact function ubiquilin serves in autophagy, particularly in the execution of macroautophagy and CMA. Is there a signal that instructs ubiquilin to choose between its known functions in autophagy and ERAD or is the choice random? What role do its different domains play in these processes? The answers to these questions are likely to be important because in previous studies we showed that overexpression of ubiquilin protects cells against potentially toxic mutant huntingtin proteins containing polyglutamine expansions. In our new work we also found that ubiquilin overexpression protects cells against starvation-induced cell death caused by mutations in presenilin-2 proteins. The underlying conclusion from these studies is that ubiquilin appears to play important roles in regulating protein degradation pathways that are likely to have important implications in cell survival. Clearly, understanding ubiquilin function in different protein degradation pathways could lead to novel approaches to prevent diseases associated with protein misfolding.

Punctum to: Rothenberg C, Srinivasan D, Mah L, Kaushik S, Peterhoff CM, Ugolino J, Fang S, Cuervo AM, Nixon RA, Monteiro MJ. Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy. Hum Mol Genet. 2010;19:3219–3232. doi: 10.1093/hmg/ddq231.