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

Figure 1.

Figure 1.

Positions of sec1 mutations. (A) Sequence conservation among the four SM protein families: Sec1, Vps33, Vps45, and Sly1. Segments of conserved amino acid sequence from S. cerevisiae Sec1p (Sce Sec1p) are aligned with the homologous sequence segments from H. sapiens Munc-18/nSec1 (Hsa nSec1), Vps33a (Hsa Vps33), Vps45 (Hsa Vps45), and Sly (Hsa Sly1). A capital letter in the consensus sequence (top line) indicates >50% identity. A lowercase letter indicates the highest probability amino acid at that position, and “x” indicates no conservation identified. The asterisks above the consensus sequence indicate conserved sites chosen for mutagenesis. SM protein sequences were separated by phylogenetics (Supplemental Figure S1). Sequences were grouped into four families (Supplemental Table 1), and each group was aligned using the CLUSTALW algorithm in the software package MacVector version 8.1.1. Alignments were evaluated to determine the consensus sequence for all four families (top line), by using the hmmbuild-f algorithm of the HMMER suite of programs (hmmer2.3.2, http://hmmer.janelia.org; Hannenhalli and Russell, 2000). (B) Conserved sites chosen for mutagenesis map to buried positions on an SM protein structure. Sites marked by asterisks in A are modeled as spheres (aspartate, red; histidine and arginine, blue) on a ribbon representation of Munc18-1/nSec1: domain 1 (aa 4-134, black), domain 2 (aa 135-245 and 480-592, light gray), and domain 3 (aa 246-479, dark gray). For labeling, amino acid numbers are based on the Munc18-1/nSec1 sequence, with the corresponding yeast numbers in parentheses. Images of nSec1 were created using Pymol (DeLano Scientific, Palo Alto, CA) and Protein Data Bank file 3C98 (Burkhardt et al., 2008), with the syntaxin 1a chain omitted. (C) class A mutations (orange spheres) map to a helical cluster in domain 3b. The positions marked are altered in the randomly generated mutants sec1-34 [G443E, K494T], sec1-38 [S391Y, D429G, E446K, I544F], sec1-39 [M402T, Y437N, I478T], and sec1-49 [Y437N, I260T] and in conserved-site mutants sec1-51 [R252A, D255A], sec1-65 [R252A, E604R], and sec1-68 [R252A, D307H, H371D]. For labeling, the amino acid numbers are based on the Munc18-1/nSec1 sequence, with the corresponding yeast numbers and mutations in parentheses. Images of nSec1 were created as described in B. (D) Many of the class B mutations (blue spheres) map to a groove between domains 1 and 2. The positions marked are altered in the randomly generated mutants sec1-32 [L417S, L639S], sec1-35 [N167I, F221Y, F235L], sec1-36 [I249N], sec1-44 [F620S, R643I, L633V, G696S], sec1-46 [F156S, I187N], sec1-48 [N81S, R638P]; in conserved-site mutants sec1-50 [E604A], sec1-52 [D255A], sec1-55 [R252L, E604L], sec1-66 [R252A, H371D], sec1-67 [D255N]; and in the groove surface mutant sec1-58 [K79E, Y80E, R169E, K170E]. Images are labeled as described in C. Where the same position has been mutated to more than one amino acid, a comma is used to separate the mutations (i.e., D255A,N). Images were created as described in B.