Abstract
When secY is overexpressed over secE or secE is underexpressed, a fraction of SecY protein is rapidly degraded in vivo. This proteolysis was unaffected in previously described protease-defective mutants examined. We found, however, that some mutations in ftsH, encoding a membrane protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family, stabilized oversynthesized SecY. This stabilization was due to a loss of FtsH function, and overproduction of the wild-type FtsH protein accelerated the degradation. The ftsH mutations also suppressed, by alleviating proteolysis of an altered form of SecY, the temperature sensitivity of the secY24 mutation, which alters SecY such that its interaction with SecE is weakened and it is destabilized at 42 degrees C. We were able to isolate a number of additional mutants with decreased ftsH expression or with an altered form of FtsH using selection/screening based on suppression of secY24 and stabilization of oversynthesized SecY. These results indicate that FtsH is required for degradation of SecY. Overproduction of SecY in the ftsH mutant cells proved to deleteriously affect cell growth and protein export, suggesting that elimination of uncomplexed SecY is important for optimum protein translocation and for the integrity of the membrane. The primary role of FtsH is discussed in light of the quite pleiotropic mutational effects, which now include stabilization of uncomplexed SecY.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akiyama Y., Ito K. Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J. 1987 Nov;6(11):3465–3470. doi: 10.1002/j.1460-2075.1987.tb02670.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Akiyama Y., Ogura T., Ito K. Involvement of FtsH in protein assembly into and through the membrane. I. Mutations that reduce retention efficiency of a cytoplasmic reporter. J Biol Chem. 1994 Feb 18;269(7):5218–5224. [PubMed] [Google Scholar]
- Akiyama Y., Shirai Y., Ito K. Involvement of FtsH in protein assembly into and through the membrane. II. Dominant mutations affecting FtsH functions. J Biol Chem. 1994 Feb 18;269(7):5225–5229. [PubMed] [Google Scholar]
- Baba T., Jacq A., Brickman E., Beckwith J., Taura T., Ueguchi C., Akiyama Y., Ito K. Characterization of cold-sensitive secY mutants of Escherichia coli. J Bacteriol. 1990 Dec;172(12):7005–7010. doi: 10.1128/jb.172.12.7005-7010.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baba T., Taura T., Shimoike T., Akiyama Y., Yoshihisa T., Ito K. A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4539–4543. doi: 10.1073/pnas.91.10.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brundage L., Hendrick J. P., Schiebel E., Driessen A. J., Wickner W. The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell. 1990 Aug 24;62(4):649–657. doi: 10.1016/0092-8674(90)90111-q. [DOI] [PubMed] [Google Scholar]
- Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
- Douville K., Leonard M., Brundage L., Nishiyama K., Tokuda H., Mizushima S., Wickner W. Band 1 subunit of Escherichia coli preportein translocase and integral membrane export factor P12 are the same protein. J Biol Chem. 1994 Jul 22;269(29):18705–18707. [PubMed] [Google Scholar]
- Dubiel W., Ferrell K., Pratt G., Rechsteiner M. Subunit 4 of the 26 S protease is a member of a novel eukaryotic ATPase family. J Biol Chem. 1992 Nov 15;267(32):22699–22702. [PubMed] [Google Scholar]
- Ghislain M., Udvardy A., Mann C. S. cerevisiae 26S protease mutants arrest cell division in G2/metaphase. Nature. 1993 Nov 25;366(6453):358–362. doi: 10.1038/366358a0. [DOI] [PubMed] [Google Scholar]
- Gottesman S., Clark W. P., de Crecy-Lagard V., Maurizi M. R. ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. Sequence and in vivo activities. J Biol Chem. 1993 Oct 25;268(30):22618–22626. [PubMed] [Google Scholar]
- Gottesman S., Maurizi M. R. Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev. 1992 Dec;56(4):592–621. doi: 10.1128/mr.56.4.592-621.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Halling S. M., Simons R. W., Way J. C., Walsh R. B., Kleckner N. DNA sequence organization of IS10-right of Tn10 and comparison with IS10-left. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2608–2612. doi: 10.1073/pnas.79.8.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herman C., Ogura T., Tomoyasu T., Hiraga S., Akiyama Y., Ito K., Thomas R., D'Ari R., Bouloc P. Cell growth and lambda phage development controlled by the same essential Escherichia coli gene, ftsH/hflB. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10861–10865. doi: 10.1073/pnas.90.22.10861. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Joly J. C., Leonard M. R., Wickner W. T. Subunit dynamics in Escherichia coli preprotein translocase. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4703–4707. doi: 10.1073/pnas.91.11.4703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Joly J. C., Wickner W. The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids. EMBO J. 1993 Jan;12(1):255–263. doi: 10.1002/j.1460-2075.1993.tb05651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kleckner N., Barker D. F., Ross D. G., Botstein D. Properties of the translocatable tetracycline-resistance element Tn10 in Escherichia coli and bacteriophage lambda. Genetics. 1978 Nov;90(3):427–461. doi: 10.1093/genetics/90.3.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunau W. H., Beyer A., Franken T., Götte K., Marzioch M., Saidowsky J., Skaletz-Rorowski A., Wiebel F. F. Two complementary approaches to study peroxisome biogenesis in Saccharomyces cerevisiae: forward and reversed genetics. Biochimie. 1993;75(3-4):209–224. doi: 10.1016/0300-9084(93)90079-8. [DOI] [PubMed] [Google Scholar]
- Matsuyama S., Akimaru J., Mizushima S. SecE-dependent overproduction of SecY in Escherichia coli. Evidence for interaction between two components of the secretory machinery. FEBS Lett. 1990 Aug 20;269(1):96–100. doi: 10.1016/0014-5793(90)81128-b. [DOI] [PubMed] [Google Scholar]
- Matsuyama S., Fujita Y., Sagara K., Mizushima S. Overproduction, purification and characterization of SecD and SecF, integral membrane components of the protein translocation machinery of Escherichia coli. Biochim Biophys Acta. 1992 Jul 13;1122(1):77–84. doi: 10.1016/0167-4838(92)90130-6. [DOI] [PubMed] [Google Scholar]
- Mhammedi-Alaoui A., Pato M., Gama M. J., Toussaint A. A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein. Mol Microbiol. 1994 Mar;11(6):1109–1116. doi: 10.1111/j.1365-2958.1994.tb00387.x. [DOI] [PubMed] [Google Scholar]
- Nakai M., Endo T., Hase T., Matsubara H. Intramitochondrial protein sorting. Isolation and characterization of the yeast MSP1 gene which belongs to a novel family of putative ATPases. J Biol Chem. 1993 Nov 15;268(32):24262–24269. [PubMed] [Google Scholar]
- Nobrega F. G., Nobrega M. P., Tzagoloff A. BCS1, a novel gene required for the expression of functional Rieske iron-sulfur protein in Saccharomyces cerevisiae. EMBO J. 1992 Nov;11(11):3821–3829. doi: 10.1002/j.1460-2075.1992.tb05474.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Osborne R. S., Silhavy T. J. PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains. EMBO J. 1993 Sep;12(9):3391–3398. doi: 10.1002/j.1460-2075.1993.tb06013.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pajic A., Tauer R., Feldmann H., Neupert W., Langer T. Yta10p is required for the ATP-dependent degradation of polypeptides in the inner membrane of mitochondria. FEBS Lett. 1994 Oct 17;353(2):201–206. doi: 10.1016/0014-5793(94)01046-3. [DOI] [PubMed] [Google Scholar]
- Pischedda C., Cocco S., Melis A., Marini M. G., Kan Y. W., Cao A., Moi P. Isolation of a differentially regulated splicing isoform of human NF-E2. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3511–3515. doi: 10.1073/pnas.92.8.3511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schatz P. J., Riggs P. D., Jacq A., Fath M. J., Beckwith J. The secE gene encodes an integral membrane protein required for protein export in Escherichia coli. Genes Dev. 1989 Jul;3(7):1035–1044. doi: 10.1101/gad.3.7.1035. [DOI] [PubMed] [Google Scholar]
- Shiba K., Ito K., Yura T., Cerretti D. P. A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant. EMBO J. 1984 Mar;3(3):631–635. doi: 10.1002/j.1460-2075.1984.tb01859.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shiba K., Ito K., Yura T. Suppressors of the secY24 mutation: identification and characterization of additional ssy genes in Escherichia coli. J Bacteriol. 1986 Jun;166(3):849–856. doi: 10.1128/jb.166.3.849-856.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sommer T., Jentsch S. A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature. 1993 Sep 9;365(6442):176–179. doi: 10.1038/365176a0. [DOI] [PubMed] [Google Scholar]
- Spong A. P., Subramani S. Cloning and characterization of PAS5: a gene required for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. J Cell Biol. 1993 Nov;123(3):535–548. doi: 10.1083/jcb.123.3.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taura T., Akiyama Y., Ito K. Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes. Mol Gen Genet. 1994 May 10;243(3):261–269. doi: 10.1007/BF00301061. [DOI] [PubMed] [Google Scholar]
- Taura T., Baba T., Akiyama Y., Ito K. Determinants of the quantity of the stable SecY complex in the Escherichia coli cell. J Bacteriol. 1993 Dec;175(24):7771–7775. doi: 10.1128/jb.175.24.7771-7775.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tokuda H. Biochemical characterization of the presecretory protein translocation machinery of Escherichia coli. FEBS Lett. 1994 Jun 6;346(1):65–68. doi: 10.1016/0014-5793(94)00317-3. [DOI] [PubMed] [Google Scholar]
- Tomoyasu T., Yuki T., Morimura S., Mori H., Yamanaka K., Niki H., Hiraga S., Ogura T. The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPases involved in membrane functions, cell cycle control, and gene expression. J Bacteriol. 1993 Mar;175(5):1344–1351. doi: 10.1128/jb.175.5.1344-1351.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Umeda M., Ohtsubo E. Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome. Gene. 1991 Feb 1;98(1):1–5. doi: 10.1016/0378-1119(91)90096-t. [DOI] [PubMed] [Google Scholar]
- Waxman L., Goldberg A. L. Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La). Science. 1986 Apr 25;232(4749):500–503. doi: 10.1126/science.2938257. [DOI] [PubMed] [Google Scholar]
- Wickner S., Gottesman S., Skowyra D., Hoskins J., McKenney K., Maurizi M. R. A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12218–12222. doi: 10.1073/pnas.91.25.12218. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson D. W., Wilcox C. A., Flynn G. C., Chen E., Kuang W. J., Henzel W. J., Block M. R., Ullrich A., Rothman J. E. A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature. 1989 Jun 1;339(6223):355–359. doi: 10.1038/339355a0. [DOI] [PubMed] [Google Scholar]