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. 2009 Nov 15;20(22):4673–4685. doi: 10.1091/mbc.E09-02-0172

Figure 4.

Figure 4.

A function distinct and before the SNARE complex binding activity of Sec1p is defective in class A mutants. (A) Reduced abundance of SNARE complexes is observed in all class A sec1 mutants. Representative Sso1p Western blots indicate the results of the assays for assembled SNARE complexes (αSec9p; top) and for Sec1p-binding to SNARE complexes (αSec1p; middle), as described in Figure 2A. Bound proteins were separated by SDS PAGE, and 0.75% of the lysates used for the IPs are shown (Input). (B) Sec9p, Sso1p, and Snc2p are present at wild-type abundance in class A sec1 mutants. Wild-type (SEC1) and sec1 mutants were lysed after 20-min incubation at the restrictive (38°C) temperature. For each lysate, 10 μg of total protein was loaded and separated by SDS-PAGE. SNARES were detected by Western blot analysis by using α-Sec9p, α-Sso1p, and α-Snc2p (Snc2-213). The sec1-65 sample was run on a separate gel, which did not resolve the doublet characteristic of Sec9p. (C) Rapid accumulation of SNARE complexes in sec18-1 increases the abundance of and binding to SNARE complexes by Sec1-34p. The results of the SNARE complex assembly (αSec9p) and binding (αSec1p) IP assays are shown for wild-type (SEC1 SEC18), sec18-1 (SEC1 sec18-1), sec1-34 (sec1-34 SEC18), and the double mutant sec1-34 sec18-1. Cells were lysed after 20-min incubation at the restrictive (38°C) temperature. The Western blot also indicates the relative abundance of Sso1p and Snc2p in 0.75% of the lysates used for the IPs, as shown in Figure 2A. (D) High-copy expression of SEC9 restores growth and partially restores SNARE complex assembly and mutant Sec1p binding to SNARE complexes in the class A mutant sec1-34. On an SC-leu-ura plate incubated at restrictive temperature (32°C), growth of the following transformants is shown in triplicate: wild-type (SEC1) with 2μ SEC9, sec1-34 with a control vector and sec1-34 with 2μ SEC9 (top). Bottom, results of the SNARE complex assembly (αSec9p) and binding (αSec1p) IP assays, performed as described in Figure 2A. Also indicated on the Western blot is the abundance of Sec9p (overexpressed in the strains transformed with 2μ SEC9), Sso1p and Sncp in 0.75% of the lysates used as inputs for the IP assays.