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. 2000 Apr 3;149(1):95–110. doi: 10.1083/jcb.149.1.95

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JCB9912051.f6a.jpg — The COOH-terminal domain of Sec2p is required for membrane attachment. (A) Truncation of the COOH terminus of Sec2p causes redistribution from the soluble to the small microsomal fraction. Cells were grown to mid-log phase, harvested, and lysed as described in the Materials and Methods. Differential centrifugation was performed on lysates from Sec2p (NY2145), Sec2-GFP (NY2146), Sec2(1-541)-GFP (NY2150), Sec2-70(1-508)-GFP (NY2148), Sec2(1-450)-GFP (NY2154), and Sec2-59(1-374)-GFP (NY2147). S0.5 supernatants were centrifuged at 10,000 g, producing the S10 and P10. The S10 supernatants were centrifuged at 100,000 g, producing S100 and P100. Equal volumes of samples were prepared for SDS-PAGE and subsequently analyzed by Western analysis with either anti-Sec2p or anti-Sec4p followed by goat anti–rabbit HRP. Upon truncation of the COOH terminus Sec2p redistributes from the soluble fraction (S100) to the microsomal pellet (P100). Hence, Sec2(1-450)-GFP, and Sec2-59(1-374)-GFP are distributed equally between the S100 and P100 while full-length Sec2p is largely soluble. (B) Sec2p membrane association is dependent on the COOH-terminal domain and this association correlates with Sec2p polarized localization. The small microsomal membrane fraction (P100, prepared as above) was resuspended and mixed at a 1:1 (vol/vol) ratio with iodixanol and centrifuged for 1 h at 625,000 g. Fractions (1–9) were collected and equal volumes were subjected to SDS-PAGE, transferred to a filter and probed with anti-GFP (Sec2 proteins, anti-Sec4p and Pma1p antibodies. Fraction 1 (and sometimes 2, top of gradient) contained the post-Golgi marker Sec4p and the plasma membrane ATPase, Pma1p (data not shown). Sec2-GFP, but not COOH-terminally truncated Sec2-59(1-374)-GFP nor the point mutant Sec2-78-GFP was enriched on membranes. (C) In addition, full-length Sec2-GFP membrane enrichment was perturbed in both sec6-4 and sec9-4 mutant backgrounds at 37°C but not the permissive temperature (25°C). This correlates with the loss of Sec2-GFP localization observed in sec6-4 at the restrictive temperature (refer to Fig. 5). As a control, Sec2-GFP was assayed in both sec3-2 and sec15-1 mutant backgrounds and found to be enriched on membranes at both temperatures. As an additional control, Sec2-GFP was assayed at 37°C in a wild-type background where it is enriched on membranes. Sec2-GFP is localized in both sec3-2 and sec15-1 backgrounds at the restrictive temperature (refer to Fig. 5).

graphic file with name JCB9912051.f6b.jpg — The COOH-terminal domain of Sec2p is required for membrane attachment. (A) Truncation of the COOH terminus of Sec2p causes redistribution from the soluble to the small microsomal fraction. Cells were grown to mid-log phase, harvested, and lysed as described in the Materials and Methods. Differential centrifugation was performed on lysates from Sec2p (NY2145), Sec2-GFP (NY2146), Sec2(1-541)-GFP (NY2150), Sec2-70(1-508)-GFP (NY2148), Sec2(1-450)-GFP (NY2154), and Sec2-59(1-374)-GFP (NY2147). S0.5 supernatants were centrifuged at 10,000 g, producing the S10 and P10. The S10 supernatants were centrifuged at 100,000 g, producing S100 and P100. Equal volumes of samples were prepared for SDS-PAGE and subsequently analyzed by Western analysis with either anti-Sec2p or anti-Sec4p followed by goat anti–rabbit HRP. Upon truncation of the COOH terminus Sec2p redistributes from the soluble fraction (S100) to the microsomal pellet (P100). Hence, Sec2(1-450)-GFP, and Sec2-59(1-374)-GFP are distributed equally between the S100 and P100 while full-length Sec2p is largely soluble. (B) Sec2p membrane association is dependent on the COOH-terminal domain and this association correlates with Sec2p polarized localization. The small microsomal membrane fraction (P100, prepared as above) was resuspended and mixed at a 1:1 (vol/vol) ratio with iodixanol and centrifuged for 1 h at 625,000 g. Fractions (1–9) were collected and equal volumes were subjected to SDS-PAGE, transferred to a filter and probed with anti-GFP (Sec2 proteins, anti-Sec4p and Pma1p antibodies. Fraction 1 (and sometimes 2, top of gradient) contained the post-Golgi marker Sec4p and the plasma membrane ATPase, Pma1p (data not shown). Sec2-GFP, but not COOH-terminally truncated Sec2-59(1-374)-GFP nor the point mutant Sec2-78-GFP was enriched on membranes. (C) In addition, full-length Sec2-GFP membrane enrichment was perturbed in both sec6-4 and sec9-4 mutant backgrounds at 37°C but not the permissive temperature (25°C). This correlates with the loss of Sec2-GFP localization observed in sec6-4 at the restrictive temperature (refer to Fig. 5). As a control, Sec2-GFP was assayed in both sec3-2 and sec15-1 mutant backgrounds and found to be enriched on membranes at both temperatures. As an additional control, Sec2-GFP was assayed at 37°C in a wild-type background where it is enriched on membranes. Sec2-GFP is localized in both sec3-2 and sec15-1 backgrounds at the restrictive temperature (refer to Fig. 5).