Protein transport by purified yeast sec complex and Kar2p without membranes

Science

Volumn 277, Issue 5328, 1997, Pages 938-941

  Matlack, Kent E S ^a   Plath, Kathrin ^a   Misselwitz, Benjamin ^a   Rapoport, Tom A ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE TRIPHOSPHATE; DETERGENT; DIGITONIN; MATING HORMONE ALPHA FACTOR; METHIONINE; SIGNAL PEPTIDE; TRITON X 100;

ARTICLE; CONTROLLED STUDY; ENDOPLASMIC RETICULUM; MEMBRANE TRANSPORT; NONHUMAN; PRIORITY JOURNAL; PROTEIN TRANSPORT; YEAST;

ADENOSINE TRIPHOSPHATE; BIOLOGICAL TRANSPORT; CROSS-LINKING REAGENTS; CYTOSOL; DETERGENTS; DIGITONIN; ENDOPLASMIC RETICULUM; FUNGAL PROTEINS; HEAT-SHOCK PROTEINS; HSP70 HEAT-SHOCK PROTEINS; LIPID BILAYERS; LIPOSOMES; MEMBRANE PROTEINS; MEMBRANE TRANSPORT PROTEINS; PROTEIN PRECURSORS; PROTEIN SORTING SIGNALS; PROTEOLIPIDS; RNA, TRANSFER; SACCHAROMYCES CEREVISIAE PROTEINS; SOLUBILITY; SUCCINIMIDES;

EID: 0030764015     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5328.938     Document Type: Article

References (26)

1. S. M. Simon and G. Blobel, Cell 65, 371 (1991); K. S. Crowley, G. D. Reinhart, A. E. Johnson, ibid. 73, 1101 (1993).
2. S. M. Simon and G. Blobel, Cell 65, 371 (1991); K. S. Crowley, G. D. Reinhart, A. E. Johnson, ibid. 73, 1101 (1993).
3. S. L. Sanders, K. M. Whitfield, J. P. Vogel, M. D. Rose, R. W. Schekman, ibid. 69, 353 (1992).
4. A. Müsch, M. Wiedmann, T. A. Rapoport, ibid., p. 343 (1992).
5. D. Gorlich and T. A. Rapoport, ibid. 75, 615 (1993).
6. W. Mothes, S. Prehn, T. A. Rapoport, EMBO J. 13, 3973 (1994).
7. D. Hanein et al., Cell 87, 721 (1996).
8. R. J. Deshaies, S. L. Sanders, D. A. Feldheim, R. Schekman, Nature 349, 806 (1991).
9. S. Panzner, L. Dreier, E. Hartmann, S. Kostka, T. A. Rapoport, Cell 81, 561 (1995).
10. I. Sadler et al., J. Cell Biol. 109, 2665 (1989); M. A. Scidmore, H. H. Okamura, M. D. Rose, Mol. Biol. Cell 4, 1145 (1993); J. L. Brodsky and R. Schekman, J. Cell Biol. 123, 1355 (1993); S. K. Lyman and R. Schekman, ibid. 131, 1163 (1995).
11. I. Sadler et al., J. Cell Biol. 109, 2665 (1989); M. A. Scidmore, H. H. Okamura, M. D. Rose, Mol. Biol. Cell 4, 1145 (1993); J. L. Brodsky and R. Schekman, J. Cell Biol. 123, 1355 (1993); S. K. Lyman and R. Schekman, ibid. 131, 1163 (1995).
12. I. Sadler et al., J. Cell Biol. 109, 2665 (1989); M. A. Scidmore, H. H. Okamura, M. D. Rose, Mol. Biol. Cell 4, 1145 (1993); J. L. Brodsky and R. Schekman, J. Cell Biol. 123, 1355 (1993); S. K. Lyman and R. Schekman, ibid. 131, 1163 (1995).
13. I. Sadler et al., J. Cell Biol. 109, 2665 (1989); M. A. Scidmore, H. H. Okamura, M. D. Rose, Mol. Biol. Cell 4, 1145 (1993); J. L. Brodsky and R. Schekman, J. Cell Biol. 123, 1355 (1993); S. K. Lyman and R. Schekman, ibid. 131, 1163 (1995).
14. P. Sanz and D. I. Meyer, J. Cell Biol. 108, 2101 (1989).
15. S. K. Lyman and R. Schekman, Cell 88, 85 (1997).
16. Purification of the Sec complex from Saccharomyces cerevisiae cells, reconstitution into proteoliposomes and in vitro translation in the reticulocyte lysate system were as described (8). Binding reaction mixture (5 or 10 μl) contained 50 mM Hepes (pH 7.5), 180 mM potassium acetate, 5 mM magnesium acetate, 2 mM dithiothreitol, 10 to 15% (v/v) glycerol, between 40 and 140 nM Sec complex in proteoliposomes, and 20% (v/v) translation mixture desalted on a NAP-5 column. After incubation at 22°C for 10 min, solubilization with 1% digitonin in 150 mM potassium acetate was on ice in 10 or 20 μl. After dilution to 130 μl under the same conditions, the samples were incubated for 4 min with μl of affinity-purified antibodies to the COOH-terminal 13 amino acids of Sec62p (8) and for 20 min with 15 μl of protein A-Sepharose. The immunoprecipitates were extensively washed with digitonin-containing buffer before analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
17. K. Finke et al., EMBO J. 15, 1482 (1996).
18. D. S. Allison and E. T. Young, Mol. Cell. Biol. 8, 1915 (1988).
19. K. E. S. Matlack, unpublished data.
20. Purified Sec complex at ∼150 nM (6 μl) in 50 mM Hepes (pH 7.5), 183 mM potassium acetate, 12% glycerol, 2 mM dithiothreitol, and 0.3% deoxyBig-CHAP (Calbiochem) was incubated on ice with phosphatidylcholine (2.7 mg/ml) and phosphatidylethanolamine (0.7 mg/ml) (Sigma). Buffer (4 μl) was added to introduce potassium acetate to 150 mM magnesium acetate to 5 mM, and digitonin to 0.5% while the lipid and deoxyBigCHAP were being diluted. Precursor (1 μl) was added, and the mixture was incubated at 22°C for 10 min before dilution and immunoprecipitation with antibodies to Sec62p.
21. Treatment with N-glycosidase F (∼24 units/ml; Boehringer) was at 0°C for 2 hours. Hot SDS sample buffer was added and samples were analyzed by immunoblotting with antibodies to Sec71p.
22. Kar2p mutants T249G, G247D, and T59G were generated by in vitro mutagenesis. Wild-type Kar2p and mutant proteins were expressed as histidine tagged proteins in Escherichia coli and purified by immobilized nickel chromatography (8). The expected properties of the mutants were confirmed (19).
23. J. Wei, J. R. Gaut, L. M. Hendershot, J. Biol. Chem. 270, 26677 (1995).
24. M. K. Nelson, T. Kurihara, P. A. Silver, Genetics 134, 159 (1993).
25. Proteolysis of ppαF:tRNA imported into proteoliposomes containing both the Sec complex and lumenal Kar2p demonstrated that, with the exception of a very short region close to the COOH-terminus, the entire protein was protected. No protected fragments were found in the absence of Kar2p and ATP.
26. We thank L. Dreier for graciously providing subcomplexes and advice; L. Hendershot for providing mammalian BiP mutants for preliminary experiments; and W. Mothes, P. Silver, B. Jungnickel, C. Shamu, and V. Siegel for critical reading of the manuscript. K.P. is supported by a grant from the MDC Berlin-Buch (Germany). Supported by NIH grant GM54238-02.