{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T08:26:36Z","timestamp":1779351996204,"version":"3.51.4"},"reference-count":26,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5328","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1997,8,15]]},"abstract":"<jats:p>Posttranslational protein translocation across the endoplasmic reticulum membrane of yeast requires a seven-component transmembrane complex (the Sec complex) in collaboration with the lumenal Kar2 protein (Kar2p). A translocation substrate was initially bound to the cytosolic face of the purified Sec complex in a signal-sequence\u2013dependent but Kar2p- and nucleotide-independent manner. In a subsequent reaction, in which Kar2p interacted with the lumenal face of the Sec complex and hydrolyzed adenosine triphosphate, the substrate moved through a channel formed by the Sec complex and was released at the lumenal end. Movement through the channel occurred in detergent solution in the absence of a lipid bilayer.<\/jats:p>","DOI":"10.1126\/science.277.5328.938","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:44:47Z","timestamp":1027763087000},"page":"938-941","source":"Crossref","is-referenced-by-count":73,"title":["Protein Transport by Purified Yeast Sec Complex and Kar2p Without Membranes"],"prefix":"10.1126","volume":"277","author":[{"given":"Kent E. S.","family":"Matlack","sequence":"first","affiliation":[{"name":"Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kathrin","family":"Plath","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Benjamin","family":"Misselwitz","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tom A.","family":"Rapoport","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(91)90455-8"},{"key":"e_1_3_1_2_3","first-page":"1101","volume":"73","author":"Crowley K. S.","year":"1993","unstructured":"Crowley K. S., Reinhart G. D., Johnson A. E., ibid. 73, 1101 (1993).","journal-title":"ibid."},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","unstructured":"S. L. Sanders K. M. Whitfield J. P. Vogel M. D. Rose R. W. Schekman ibid. 69 353 (1992).","DOI":"10.1016\/0092-8674(92)90415-9"},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","unstructured":"A. M\u00fcsch M. Wiedmann T. A. Rapoport ibid. p. 343 (1992).","DOI":"10.1016\/0092-8674(92)90414-8"},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","unstructured":"D. Gorlich and T. A. Rapoport ibid. 75 615 (1993).","DOI":"10.1016\/0092-8674(93)90483-7"},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"3973","DOI":"10.1002\/j.1460-2075.1994.tb06713.x","volume":"13","author":"Mothes W.","year":"1994","unstructured":"Mothes W., Prehn S., Rapoport T. A., EMBO J. 13, 3973 (1994).","journal-title":"EMBO J."},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1016\/S0092-8674(00)81391-4","volume":"87","author":"Hanein D.","year":"1996","unstructured":"Hanein D., et al., Cell 87, 721 (1996).","journal-title":"Cell"},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1038\/349806a0","volume":"349","author":"Deshaies R. J.","year":"1991","unstructured":"Deshaies R. J., Sanders S. L., Feldheim D. A., Schekman R., Nature 349, 806 (1991).","journal-title":"Nature"},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1016\/0092-8674(95)90077-2","volume":"81","author":"Panzner S.","year":"1995","unstructured":"Panzner S., Dreier L., Hartmann E., Kostka S., Rapoport T. A., Cell 81, 561 (1995).","journal-title":"Cell"},{"key":"e_1_3_1_10_2","doi-asserted-by":"crossref","first-page":"2665","DOI":"10.1083\/jcb.109.6.2665","volume":"109","author":"Sadler I.","year":"1989","unstructured":"Sadler I., et al., J. Cell Biol. 109, 2665 (1989);","journal-title":"J. Cell Biol."},{"key":"e_1_3_1_10_3","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.1091\/mbc.4.11.1145","volume":"4","author":"Scidmore M. A.","year":"1993","unstructured":"Scidmore M. A., Okamura H. H., Rose M. D., Mol. Biol. Cell 4, 1145 (1993);","journal-title":"Mol. Biol. Cell"},{"key":"e_1_3_1_10_4","doi-asserted-by":"crossref","first-page":"1355","DOI":"10.1083\/jcb.123.6.1355","volume":"123","author":"Brodsky J. L.","year":"1993","unstructured":"Brodsky J. L., Schekman R., J. Cell Biol. 123, 1355 (1993) ;","journal-title":"J. Cell Biol."},{"key":"e_1_3_1_10_5","first-page":"1163","volume":"131","author":"Lyman S. K.","year":"1995","unstructured":"Lyman S. K., Schekman R., ibid. 131, 1163 (1995).","journal-title":"ibid."},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","first-page":"2101","DOI":"10.1083\/jcb.108.6.2101","volume":"108","author":"Sanz P.","year":"1989","unstructured":"Sanz P., Meyer D. I., J. Cell Biol. 108, 2101 (1989).","journal-title":"J. Cell Biol."},{"key":"e_1_3_1_12_2","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/S0092-8674(00)81861-9","volume":"88","author":"Lyman S. K.","year":"1997","unstructured":"Lyman S. K., Schekman R., Cell 88, 85 (1997).","journal-title":"Cell"},{"key":"e_1_3_1_13_2","unstructured":"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 \u03bcl) 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\u00b0C for 10 min solubilization with 1% digitonin in 150 mM potassium acetate was on ice in 10 or 20 \u03bcl. After dilution to 130 \u03bcl under the same conditions the samples were incubated for 40 min with 1 \u03bcl of affinity-purified antibodies to the COOH-terminal 13 amino acids of Sec62p (8) and for 20 min with 15 \u03bcl of protein A-Sepharose. The immunoprecipitates were extensively washed with digitonin-containing buffer before analysis by SDS\u2013polyacrylamide gel electrophoresis (SDS-PAGE)."},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","first-page":"1482","DOI":"10.1002\/j.1460-2075.1996.tb00492.x","volume":"15","author":"Finke K.","year":"1996","unstructured":"Finke K., et al., EMBO J. 15, 1482 (1996).","journal-title":"EMBO J."},{"key":"e_1_3_1_15_2","first-page":"1915","volume":"8","author":"Allison D. S.","year":"1988","unstructured":"Allison D. S., Young E. T., Mol. Cell. Biol. 8, 1915 (1988).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_16_2","unstructured":"K. E. S. Matlack unpublished data."},{"key":"e_1_3_1_17_2","unstructured":"Purified Sec complex at \u223c150 nM (6 \u03bcl) in 50 mM Hepes (pH 7.5) 183 mM potassium acetate 12% glycerol 2 mM dithiothreitol and 0.3% deoxyBigCHAP (Calbiochem) was incubated on ice with phosphatidylcholine (2.7 mg\/ml) and phosphatidylethanolamine (0.7 mg\/ml) (Sigma). Buffer (4 \u03bcl) 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 \u03bcl) was added and the mixture was incubated at 22\u00b0C for 10 min before dilution and immunoprecipitation with antibodies to Sec62p."},{"key":"e_1_3_1_18_2","unstructured":"Treatment with N-glycosidase F (\u223c24 units\/ml; Boehringer) was at 0\u00b0C for 2 hours. Hot SDS sample buffer was added and samples were analyzed by immunoblotting with antibodies to Sec71p."},{"key":"e_1_3_1_19_2","unstructured":"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)."},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","first-page":"26677","DOI":"10.1074\/jbc.270.44.26677","volume":"270","author":"Wei J.","year":"1995","unstructured":"Wei J., Gaut J. R., Hendershot L. M., J. Biol. Chem. 270, 26677 (1995).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_21_2","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1093\/genetics\/134.1.159","volume":"134","author":"Nelson M. K.","year":"1993","unstructured":"Nelson M. K., Kurihara T., Silver P. A., Genetics 134, 159 (1993).","journal-title":"Genetics"},{"key":"e_1_3_1_22_2","unstructured":"Proteolysis of pp\u03b1F: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."},{"key":"e_1_3_1_23_2","unstructured":"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."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.277.5328.938","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T05:47:00Z","timestamp":1705124820000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.277.5328.938"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1997,8,15]]},"references-count":26,"journal-issue":{"issue":"5328","published-print":{"date-parts":[[1997,8,15]]}},"alternative-id":["10.1126\/science.277.5328.938"],"URL":"https:\/\/doi.org\/10.1126\/science.277.5328.938","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1997,8,15]]}}}