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. 1995 Dec 2;131(6):1377–1386. doi: 10.1083/jcb.131.6.1377

Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation

PMCID: PMC2120672  PMID: 8522598

Abstract

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro- alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.

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Selected References

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  1. Aquila H., Link T. A., Klingenberg M. The uncoupling protein from brown fat mitochondria is related to the mitochondrial ADP/ATP carrier. Analysis of sequence homologies and of folding of the protein in the membrane. EMBO J. 1985 Sep;4(9):2369–2376. doi: 10.1002/j.1460-2075.1985.tb03941.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bankaitis V. A., Aitken J. R., Cleves A. E., Dowhan W. An essential role for a phospholipid transfer protein in yeast Golgi function. Nature. 1990 Oct 11;347(6293):561–562. doi: 10.1038/347561a0. [DOI] [PubMed] [Google Scholar]
  3. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  4. 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]
  5. Capasso J. M., Keenan T. W., Abeijon C., Hirschberg C. B. Mechanism of phosphorylation in the lumen of the Golgi apparatus. Translocation of adenosine 5'-triphosphate into Golgi vesicles from rat liver and mammary gland. J Biol Chem. 1989 Mar 25;264(9):5233–5240. [PubMed] [Google Scholar]
  6. Clairmont C. A., De Maio A., Hirschberg C. B. Translocation of ATP into the lumen of rough endoplasmic reticulum-derived vesicles and its binding to luminal proteins including BiP (GRP 78) and GRP 94. J Biol Chem. 1992 Feb 25;267(6):3983–3990. [PubMed] [Google Scholar]
  7. Cleves A. E., Novick P. J., Bankaitis V. A. Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function. J Cell Biol. 1989 Dec;109(6 Pt 1):2939–2950. doi: 10.1083/jcb.109.6.2939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Esnault Y., Feldheim D., Blondel M. O., Schekman R., Képès F. SSS1 encodes a stabilizing component of the Sec61 subcomplex of the yeast protein translocation apparatus. J Biol Chem. 1994 Nov 4;269(44):27478–27485. [PubMed] [Google Scholar]
  9. Georgopoulos C., Welch W. J. Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol. 1993;9:601–634. doi: 10.1146/annurev.cb.09.110193.003125. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Glick B. S. Can Hsp70 proteins act as force-generating motors? Cell. 1995 Jan 13;80(1):11–14. doi: 10.1016/0092-8674(95)90444-1. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Hemmings B. A., Zubenko G. S., Hasilik A., Jones E. W. Mutant defective in processing of an enzyme located in the lysosome-like vacuole of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Jan;78(1):435–439. doi: 10.1073/pnas.78.1.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klingenberg M. Mitochondrial carrier family: ADP/ATP carrier as a carrier paradigm. Soc Gen Physiol Ser. 1993;48:201–212. [PubMed] [Google Scholar]
  16. Krämer R., Heberger C. Functional reconstitution of carrier proteins by removal of detergent with a hydrophobic ion exchange column. Biochim Biophys Acta. 1986 Dec 16;863(2):289–296. doi: 10.1016/0005-2736(86)90269-5. [DOI] [PubMed] [Google Scholar]
  17. Krämer R., Klingenberg M. Reconstitution of adenine nucleotide transport from beef heart mitochondria. Biochemistry. 1979 Sep 18;18(19):4209–4215. doi: 10.1021/bi00586a027. [DOI] [PubMed] [Google Scholar]
  18. Mandon E. C., Milla M. E., Kempner E., Hirschberg C. B. Purification of the Golgi adenosine 3'-phosphate 5'-phosphosulfate transporter, a homodimer within the membrane. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10707–10711. doi: 10.1073/pnas.91.22.10707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mayinger P., Meyer D. I. An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum. EMBO J. 1993 Feb;12(2):659–666. doi: 10.1002/j.1460-2075.1993.tb05699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McGee T. P., Skinner H. B., Whitters E. A., Henry S. A., Bankaitis V. A. A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes. J Cell Biol. 1994 Feb;124(3):273–287. doi: 10.1083/jcb.124.3.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Novick P., Osmond B. C., Botstein D. Suppressors of yeast actin mutations. Genetics. 1989 Apr;121(4):659–674. doi: 10.1093/genetics/121.4.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Palmieri F., Indiveri C., Bisaccia F., Krämer R. Functional properties of purified and reconstituted mitochondrial metabolite carriers. J Bioenerg Biomembr. 1993 Oct;25(5):525–535. doi: 10.1007/BF01108409. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Pollard T. D. Actin. Curr Opin Cell Biol. 1990 Feb;2(1):33–40. doi: 10.1016/s0955-0674(05)80028-6. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Rothblatt J. A., Meyer D. I. Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system. Cell. 1986 Feb 28;44(4):619–628. doi: 10.1016/0092-8674(86)90271-0. [DOI] [PubMed] [Google Scholar]
  31. Sadis S., Hightower L. E. Unfolded proteins stimulate molecular chaperone Hsc70 ATPase by accelerating ADP/ATP exchange. Biochemistry. 1992 Oct 6;31(39):9406–9412. doi: 10.1021/bi00154a012. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Sanz P., Meyer D. I. Secretion in yeast: preprotein binding to a membrane receptor and ATP-dependent translocation are sequential and separable events in vitro. J Cell Biol. 1989 Jun;108(6):2101–2106. doi: 10.1083/jcb.108.6.2101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sanz P., Meyer D. I. Signal recognition particle (SRP) stabilizes the translocation-competent conformation of pre-secretory proteins. EMBO J. 1988 Nov;7(11):3553–3557. doi: 10.1002/j.1460-2075.1988.tb03232.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schekman R. Translocation gets a push. Cell. 1994 Sep 23;78(6):911–913. doi: 10.1016/0092-8674(94)90265-8. [DOI] [PubMed] [Google Scholar]
  36. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Simons J. F., Ferro-Novick S., Rose M. D., Helenius A. BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast. J Cell Biol. 1995 Jul;130(1):41–49. doi: 10.1083/jcb.130.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stevens T., Esmon B., Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell. 1982 Sep;30(2):439–448. doi: 10.1016/0092-8674(82)90241-0. [DOI] [PubMed] [Google Scholar]
  40. Toyn J., Hibbs A. R., Sanz P., Crowe J., Meyer D. I. In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation. EMBO J. 1988 Dec 20;7(13):4347–4353. doi: 10.1002/j.1460-2075.1988.tb03333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Whitters E. A., Cleves A. E., McGee T. P., Skinner H. B., Bankaitis V. A. SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast. J Cell Biol. 1993 Jul;122(1):79–94. doi: 10.1083/jcb.122.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]

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