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. 1998 Jun 1;141(5):1107-19.
doi: 10.1083/jcb.141.5.1107.

Sec35p, a novel peripheral membrane protein, is required for ER to Golgi vesicle docking

S M VanRheenen  1 , X Cao, V V Lupashin, C Barlowe, M G Waters
Affiliations

Sec35p, a novel peripheral membrane protein, is required for ER to Golgi vesicle docking

S M VanRheenen et al. J Cell Biol. .

Abstract

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (. Genetics. 142:393-406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE-associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.

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Figures

Figure 1
Figure 1
Predicted amino acid sequence of Sec35p and Sec35-1p. (a) Protein sequence of Sec35p. SEC35 was sequenced as part of the Yeast Genome Sequencing project and corresponds to ORF YGR120c, GenBank/EMBL/DDBJ accession number Z72905. (b) Sequence of wild-type SEC35 from bp 613 to 628 with the transversion mutation at bp 624 (t to a) and corresponding codon change (tyrosine to ochre) present in the sec35-1 allele indicated with arrows.
Figure 2
Figure 2
A haploid strain containing a deletion of SEC35 exhibits a severe growth defect at 30°C and is inviable at 21°C; the cold-sensitive lethality can be rescued by the expression of SEC35, low levels of SLY1-20, or high levels of YPT1. The sec35Δ/SEC35 heterozygous deletion strain alone (a and b), or transformed with pSEC35 (CEN) (c), pYPT1 (2μm) (d), or pSLY1-20 (CEN) (e) was sporulated and dissected. The resulting tetrads were incubated at 30°C for 7 d (a) or at 21°C for 5 d (b and c) or 7 d (d and e) on YPD plates. In c–e, only tetrads containing four viable spores are shown; tetrads with two or three viable spores were assumed to result from tetrads in which one or both of the sec35Δ haploid spores failed to receive the suppressing plasmid.
Figure 3
Figure 3
Suppression of the temperature-sensitive growth phenotype of the sec35-1 strain. Wild-type and sec35-1 strains containing the indicated plasmids were grown to stationary phase in synthetic media and used for a series of 10-fold dilutions. The stationary phase culture and five serial dilutions of each strain were spotted onto YPD plates and grown for 4 d at 37.5°C.
Figure 6
Figure 6
Characterization of the affinity-purified anti-Sec35p antibody. Extracts were made by glass bead lysis from logarithmically growing strains: wild-type, sec35Δ transformed with pSLY1-20 (CEN), wild-type transformed with pSEC35 (2μm), and sec35-1. All strains were incubated at 21°C before lysis except for the sec35-1 strain, which was maintained at 21°C or shifted to 38°C for 1 h before lysis. Extracts (0.2 OD595 per lane) were separated by SDS-12%PAGE and immunoblotted with affinity-purified anti-Sec35p antibodies. The migration of mol wt markers is shown on the right.
Figure 4
Figure 4
Suppression of the ER to Golgi transport defect of sec35-1 by expression of SLY1-20 or high levels of YPT1. Wild-type and sec18-1 strains, as well as the sec35-1 strain with and without the indicated plasmids, were grown at 21°C to mid-logarithmic phase and then shifted to 39°C for 45 min. Cells were labeled at 39°C for 10 min with Tran35S-label and then chased for 20 min at the same temperature. CPY was immunoprecipitated from each strain, resolved by SDS-7%PAGE, and then visualized by autoradiography. The migration of the ER precursor (p1) and mature (m) forms of CPY are indicated on the left.
Figure 5
Figure 5
The sec35-1 allele exhibits a synthetic interaction with ypt1-3 and uso1-1. The sec35-1 strain was mated to a strain bearing the ypt1-3 allele (a) or the uso1-1 allele (b), and the resulting diploid strains were sporulated, dissected on YPD plates, and then incubated at 30°C for 3 d. Six representative tetrads of each dissection are shown.
Figure 7
Figure 7
Sec35p is a peripheral membrane protein. (a) Sec35p partitions into soluble and membrane-associated pools. A wild-type strain was grown to logarithmic phase, lysed with glass beads, and then centrifuged at 1,000 g for 3 min to generate the S1 supernatant fraction. The S1 supernatant was centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. (b) The membrane-associated portion of Sec35p fractionates similarly to the Golgi protein, Sed5p. The S1 supernatant was centrifuged at 10,000 g to generate the S10 supernatant and P10 pellet fractions, and the S10 was subsequently centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. (c) Sec35p behaves as a peripheral membrane protein. The S1 supernatant was incubated with buffer 88, 1% Triton X-100, 1 M NaCl, or 100 mM Na2CO3, pH 11, centrifuged at 175,000 g, and separated into supernatant and pellet fractions. (a–c) Aliquots of each fraction were separated by SDS-12%PAGE and immunoblotted with affinity-purified anti-Sec35p (top panel), anti-Sed5p (center panel), or anti-PGK (bottom panel) antibodies. The samples loaded in each lane were derived from equivalent amounts of starting material.
Figure 8
Figure 8
Sec35p is required for ER to Golgi transport in vitro. Semi-intact yeast cell membranes prepared from wild-type (A) or sec35-1 (B) strains were incubated at 23° or 29°C for 60 min. Reactions contained an ATP regeneration system alone (No addition, solid bars), COPII, Uso1p, and LMA1 (Reconstituted, open bars), or COPII, Uso1p, LMA1 and His6-Sec35p (Reconstituted + Sec35p, hatched bars). Outer-chain modified forms of [35S]gp-α-factor were immunoprecipitated with anti–α1,6-mannose–specific antibodies and the percent transport reflects the amount of total [35S]gp-α-factor that has acquired Golgi-specific outer-chain modifications.
Figure 9
Figure 9
Sec35p is required for vesicle docking. Semi-intact yeast cell membranes prepared from wild-type or sec35-1 strains were incubated at 23° or 29°C for 30 min. Reactions contained an ATP regeneration system alone (No addition, solid bars), COPII (COPII, wide hatched bars), COPII and Uso1p (COPII + Uso1p, open bars), or COPII, Uso1p, and His6-Sec35p (COPII, Uso1p + Sec35p, narrow hatched bars). Freely diffusible vesicles containing [35S]gp-α-factor were separated from semi-intact membranes by centrifugation at 18,000 g for 3 min. The percent diffusible vesicles reflects the amount of total concanavalin A precipitable [35S]gp-α-factor contained in the 18,000 g supernatant fluid.
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