The Saccharomyces cerevisiae SCS2 gene product, a homolog of a synaptobrevin-associated protein, is an integral membrane protein of the endoplasmic reticulum and is required for inositol metabolism - PubMed (original) (raw)

The Saccharomyces cerevisiae SCS2 gene product, a homolog of a synaptobrevin-associated protein, is an integral membrane protein of the endoplasmic reticulum and is required for inositol metabolism

S Kagiwada et al. J Bacteriol. 1998 Apr.

Abstract

The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 and hac1/ire15 mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39-45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from Aplysia californica (P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580-1583, 1995). In this study we have characterized an SCS2 gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34 degrees C. Genetic studies reveal that the overexpression of the INO1 gene rescues the inositol auxotrophy of the SCS2 disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the SCS2 disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.

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Figures

FIG. 1

FIG. 1

Inositol auxotrophy of _scs2_Δ strains. KY360 (_scs2_Δ::TRP1) cells transformed with YCp_lac_33 (control), YCp(SCS2), or YCp(HA-SCS2) were streaked for isolation on either inositol-containing (INO+) or inositol-free (INO−) minimal medium and incubated at 34°C for 72 h or at 37°C for 96 h.

FIG. 2

FIG. 2

Inositol auxotrophy of _scs2_Δ strains transformed with INO1 or INO2. KY360 (_scs2_Δ::TRP1) cells transformed with YCp_lac_33 [vector control for YCp(INO1)], YEplac195 [vector control for YEp(INO2)], YCp(INO1), or YEp(INO2) were streaked for isolation on either inositol-containing (INO+), or inositol-free (INO−) minimal medium and incubated at 34°C for 72 h.

FIG. 3

FIG. 3

Characterization of Scs2p by Western blotting. (A) Strains were grown in minimal medium, converted to spheroplasts, and lysed osmotically. The resultant lysate was centrifuged at 800 × g to yield LSS. The LSS was separated by SDS–10% PAGE and immunoblotted with the anti-Scs2p polyclonal antibody (α-Scs2p) (lanes 1 to 3) or the anti-HA monoclonal antibody (α-HA) (lanes 4 and 5). The strains employed were YPH500 (wild type [WT]) (lane 1) and KY360 (_scs2_Δ::TRP1) harboring either YCp_lac_33 (lanes 2 and 4) or YCp(HA-SCS2) (lanes 3 and 5). (B) The LSS fraction of wild-type cells (CTY182) was subjected to two rounds of differential centrifugation to yield 12,000 × g pellet and supernatant fractions (12P and 12S, respectively) and 100,000 × g pellet and supernatant fractions (100P and 100S, respectively). These fractions were resolved by SDS–10% PAGE and immunoblotted for the presence of Scs2p and for markers of the ER membrane (Dpm1p), ER lumen (Kar2p), and cytoplasm/Golgi membrane (Sec14p). (C) The 12P fraction of wild-type cells (CTY182) was incubated with 0.2 volume of one of the following solutions: H2O (control), 5 M KCl, 10 M urea, 0.5 M Na2CO3 (pH 11), or 5% Triton X-100 (TX-100). After incubation at 4°C for 30 min, samples were centrifuged at 100,000 × g for 30 min to separate supernatant (S) and pellet (P) fractions. These fractions were resolved by SDS–10% PAGE and immunoblotted with the anti-Scs2p polyclonal antibody. (D) The 12P fraction of KY360 (_scs2_Δ::TRP1) harboring YCp(HA-SCS2) was incubated with 0.1 mg of trypsin per ml in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 0.1% Triton X-100 (TX-100). Soybean trypsin inhibitor (STI) was added at the beginning (lanes 3 and 4) or end (lanes 1 and 2) of the incubation. Samples were resolved by SDS–10% PAGE and immunoblotted with the anti-HA monoclonal antibody (top panel) or the anti-Kar2p polyclonal antibody (bottom panel). In panels A and C, the numbers on the left are molecular masses in kilodaltons.

FIG. 4

FIG. 4

Localization of Scs2p. Indirect immunofluorescence was carried out with YPH500 cells (wild type) (a, b, and c), KY360 cells (_scs2_Δ::TRP1) (d, e, and f), and KY360 cells harboring YCp(HA-SCS2) (g, h, and i). The cells in panels a to f were incubated with the rabbit anti-Scs2p polyclonal antibody and the mouse anti-Dpm1p monoclonal antibody, and cells in panels g to i were incubated with the rabbit anti-HA polyclonal antibody and the mouse anti-Dpm1p monoclonal antibody. Samples were stained with tetramethylrhodamine isothiocyanate-conjugated anti-rabbit IgG to detect Scs2p (a, d, and g), fluorescein isothiocyanate-conjugated anti-mouse IgG to detect Dpm1p (b, e, and h), and DAPI to indicate the position of the nuclei (c, f, and i).

FIG. 5

FIG. 5

The 16-amino-acid conserved region is required for Scs2p activity. (A) Alignment of a 16-amino-acid sequence of Scs2p with those of S. pombe, A. californica (VAP-33), and mouse and human homologs. The sequences of the mouse and human homologs are deduced from nucleotide sequences of expressed sequence tags. Residues identical in all five sequences are boxed. Homologous sequences of MSP1A, TER ATPase, and Cdc48p are also shown. For Scs2p, the S. pombe homolog, VAP-33, MSP1A, TER ATPase, and Cdc48p, the numbers refer to amino acid positions. GenBank accession numbers are D44493 (Scs2p), Z73099 (S. pombe), U36779 (VAP-33), W54842 (mouse), N34715 (human), P53021 (MSP1A), U11760 (TER ATPase), and X56956 (Cdc48p). (B) Overproduction of Scs2pΔ36-53 failed to suppress the inositol auxotrophy of the _scs2_Δ strain. KY360 (_scs2_Δ::TRP1) cells transformed with YEp_lac_195, YEp(SCS2), YEp(_SCS2_Δ36-53), YCp_lac_33, YCp(SCS2), or YCp(_SCS2_Δ36-53) were streaked for isolation on either inositol-containing (INO+) or inositol-free (INO−) minimal medium and incubated at 34°C for 72 h or at 37°C for 96 h.

FIG. 6

FIG. 6

Disruption of SCS2 has no effect on protein secretion. (A) Sugar modification of invertase. Cells were grown in YPD medium at 25°C and shifted to YP medium with 0.1% glucose. After 2 h of incubation at 30°C, cells were converted to spheroplasts and separated into the intracellular (I) and extracellular (E) fractions. These fractions were subjected to native gel activity staining for invertase. The position of nonglycosylated (cytosolic) invertase is indicated by an arrow. Strains used were CTY182 (wild type [WT]) (lanes 1 and 2) and KY356 (_scs2_Δ::URA3) (lanes 3 and 4). (B) Halo assay for α-factor production. YPH500 (WT) and KY360 (_scs2_Δ::TRP1) cells were spotted on a lawn of a-mating-type cells (MATa sst2) on a plate and incubated at 30°C for 48 h to allow halos to develop.

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