Abstract
Two interacting heat shock cognate proteins in the lumen of the yeast endoplasmic reticulum (ER), Sec63p and BiP (Kar2p), are required for posttranslational translocation of yeast alpha-factor precursor in vitro. To investigate the role of these proteins in cotranslational translocation, we examined the import of invertase into wild-type, sec63, and kar2 mutant yeast membranes. We found that Sec63p and Kar2p are necessary for both co- and posttranslational translocation in yeast. Several kar2 mutants, one of which had normal ATPase activity, were defective in cotranslational translocation of invertase. We conclude that the requirement for BiP/Kar2p, which is not seen in a reaction reconstituted with pure mammalian membrane proteins [Görlich, D. & Rapoport, T.A. (1993) Cell 75, 615-630], is not due to a distinction between cotranslational translocation in mammalian cells and posttranslational translocation in yeast cells.
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- Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brodsky J. L., Hamamoto S., Feldheim D., Schekman R. Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70. J Cell Biol. 1993 Jan;120(1):95–102. doi: 10.1083/jcb.120.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brodsky J. L., Schekman R. A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome. J Cell Biol. 1993 Dec;123(6 Pt 1):1355–1363. doi: 10.1083/jcb.123.6.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brown J. D., Hann B. C., Medzihradszky K. F., Niwa M., Burlingame A. L., Walter P. Subunits of the Saccharomyces cerevisiae signal recognition particle required for its functional expression. EMBO J. 1994 Sep 15;13(18):4390–4400. doi: 10.1002/j.1460-2075.1994.tb06759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
- Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
- Deshaies R. J., Schekman R. SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum. J Cell Biol. 1989 Dec;109(6 Pt 1):2653–2664. doi: 10.1083/jcb.109.6.2653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feldheim D., Rothblatt J., Schekman R. Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation. Mol Cell Biol. 1992 Jul;12(7):3288–3296. doi: 10.1128/mcb.12.7.3288. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Felici F., Cesareni G., Hughes J. M. The most abundant small cytoplasmic RNA of Saccharomyces cerevisiae has an important function required for normal cell growth. Mol Cell Biol. 1989 Aug;9(8):3260–3268. doi: 10.1128/mcb.9.8.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Flynn G. C., Chappell T. G., Rothman J. E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989 Jul 28;245(4916):385–390. doi: 10.1126/science.2756425. [DOI] [PubMed] [Google Scholar]
- Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
- Görlich D., Rapoport T. A. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell. 1993 Nov 19;75(4):615–630. doi: 10.1016/0092-8674(93)90483-7. [DOI] [PubMed] [Google Scholar]
- Haas I. G., Wabl M. Immunoglobulin heavy chain binding protein. Nature. 1983 Nov 24;306(5941):387–389. doi: 10.1038/306387a0. [DOI] [PubMed] [Google Scholar]
- Hann B. C., Walter P. The signal recognition particle in S. cerevisiae. Cell. 1991 Oct 4;67(1):131–144. doi: 10.1016/0092-8674(91)90577-l. [DOI] [PubMed] [Google Scholar]
- Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
- Hansen W., Walter P. Prepro-carboxypeptidase Y and a truncated form of pre-invertase, but not full-length pre-invertase, can be posttranslationally translocated across microsomal vesicle membranes from Saccharomyces cerevisiae. J Cell Biol. 1988 Apr;106(4):1075–1081. doi: 10.1083/jcb.106.4.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kronidou N. G., Oppliger W., Bolliger L., Hannavy K., Glick B. S., Schatz G., Horst M. Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12818–12822. doi: 10.1073/pnas.91.26.12818. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller J. D., Tajima S., Lauffer L., Walter P. The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane. J Cell Biol. 1995 Feb;128(3):273–282. doi: 10.1083/jcb.128.3.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nelson M. K., Kurihara T., Silver P. A. Extragenic suppressors of mutations in the cytoplasmic C terminus of SEC63 define five genes in Saccharomyces cerevisiae. Genetics. 1993 May;134(1):159–173. doi: 10.1093/genetics/134.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nguyen T. H., Law D. T., Williams D. B. Binding protein BiP is required for translocation of secretory proteins into the endoplasmic reticulum in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1565–1569. doi: 10.1073/pnas.88.4.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nicchitta C. V., Blobel G. Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation. Cell. 1993 Jun 4;73(5):989–998. doi: 10.1016/0092-8674(93)90276-v. [DOI] [PubMed] [Google Scholar]
- Normington K., Kohno K., Kozutsumi Y., Gething M. J., Sambrook J. S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell. 1989 Jun 30;57(7):1223–1236. doi: 10.1016/0092-8674(89)90059-7. [DOI] [PubMed] [Google Scholar]
- Nunnari J., Walter P. Protein targeting to and translocation across the membrane of the endoplasmic reticulum. Curr Opin Cell Biol. 1992 Aug;4(4):573–580. doi: 10.1016/0955-0674(92)90074-m. [DOI] [PubMed] [Google Scholar]
- Ogg S. C., Poritz M. A., Walter P. Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae. Mol Biol Cell. 1992 Aug;3(8):895–911. doi: 10.1091/mbc.3.8.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Panzner S., Dreier L., Hartmann E., Kostka S., Rapoport T. A. Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p. Cell. 1995 May 19;81(4):561–570. doi: 10.1016/0092-8674(95)90077-2. [DOI] [PubMed] [Google Scholar]
- Rapoport T. A. Transport of proteins across the endoplasmic reticulum membrane. Science. 1992 Nov 6;258(5084):931–936. doi: 10.1126/science.1332192. [DOI] [PubMed] [Google Scholar]
- Rassow J., Maarse A. C., Krainer E., Kübrich M., Müller H., Meijer M., Craig E. A., Pfanner N. Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44. J Cell Biol. 1994 Dec;127(6 Pt 1):1547–1556. doi: 10.1083/jcb.127.6.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rose M. D., Misra L. M., Vogel J. P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell. 1989 Jun 30;57(7):1211–1221. doi: 10.1016/0092-8674(89)90058-5. [DOI] [PubMed] [Google Scholar]
- Rothblatt J. A., Deshaies R. J., Sanders S. L., Daum G., Schekman R. Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast. J Cell Biol. 1989 Dec;109(6 Pt 1):2641–2652. doi: 10.1083/jcb.109.6.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothblatt J. A., Meyer D. I. Secretion in yeast: translocation and glycosylation of prepro-alpha-factor in vitro can occur via an ATP-dependent post-translational mechanism. EMBO J. 1986 May;5(5):1031–1036. doi: 10.1002/j.1460-2075.1986.tb04318.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothblatt J. A., Webb J. R., Ammerer G., Meyer D. I. Secretion in yeast: structural features influencing the post-translational translocation of prepro-alpha-factor in vitro. EMBO J. 1987 Nov;6(11):3455–3463. doi: 10.1002/j.1460-2075.1987.tb02669.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sadler I., Chiang A., Kurihara T., Rothblatt J., Way J., Silver P. A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein. J Cell Biol. 1989 Dec;109(6 Pt 1):2665–2675. doi: 10.1083/jcb.109.6.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanders S. L., Schekman R. Polypeptide translocation across the endoplasmic reticulum membrane. J Biol Chem. 1992 Jul 15;267(20):13791–13794. [PubMed] [Google Scholar]
- Sanders S. L., Whitfield K. M., Vogel J. P., Rose M. D., Schekman R. W. Sec61p and BiP directly facilitate polypeptide translocation into the ER. Cell. 1992 Apr 17;69(2):353–365. doi: 10.1016/0092-8674(92)90415-9. [DOI] [PubMed] [Google Scholar]
- Schneider H. C., Berthold J., Bauer M. F., Dietmeier K., Guiard B., Brunner M., Neupert W. Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature. 1994 Oct 27;371(6500):768–774. doi: 10.1038/371768a0. [DOI] [PubMed] [Google Scholar]
- Scidmore M. A., Okamura H. H., Rose M. D. Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum. Mol Biol Cell. 1993 Nov;4(11):1145–1159. doi: 10.1091/mbc.4.11.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simon S. M., Peskin C. S., Oster G. F. What drives the translocation of proteins? Proc Natl Acad Sci U S A. 1992 May 1;89(9):3770–3774. doi: 10.1073/pnas.89.9.3770. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stirling C. J., Hewitt E. W. The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19. Nature. 1992 Apr 9;356(6369):534–537. doi: 10.1038/356534a0. [DOI] [PubMed] [Google Scholar]
- Taussig R., Carlson M. Nucleotide sequence of the yeast SUC2 gene for invertase. Nucleic Acids Res. 1983 Mar 25;11(6):1943–1954. doi: 10.1093/nar/11.6.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Verner K., Schatz G. Protein translocation across membranes. Science. 1988 Sep 9;241(4871):1307–1313. doi: 10.1126/science.2842866. [DOI] [PubMed] [Google Scholar]
- Vogel J. P., Misra L. M., Rose M. D. Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J Cell Biol. 1990 Jun;110(6):1885–1895. doi: 10.1083/jcb.110.6.1885. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Waters M. G., Blobel G. Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis. J Cell Biol. 1986 May;102(5):1543–1550. doi: 10.1083/jcb.102.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]