The Snc v-SNAREs interact physically with Gcs1. (A) Snc2 interacts with Gcs1, as assayed using the two-hybrid lacZ detection assay. Yeast (Y153) were transformed with a prey plasmid expressing Snc2 fused to the transactivating domain of Gal4 (Snc2^8-115; plasmid pPP381-39) and bait plasmids, including vectors expressing the DNA-binding domain of Gal4 alone (-; plasmid pGBT9), or fused with Snf1 (Snf1; plasmid pSE1112) or Gcs1 (Gcs1; plasmid pPP269). Cells were grown in patches on selective medium, replica plated onto nitrocellulose filters, lysed in liquid nitrogen, and measured for β-galactosidase activity using standard techniques. (B) Snc1 and 2 interact with Gcs1, as assayed using the two-hybrid 3-AT growth assay. Bait plasmids expressing the Gal4-transactivating domain fused to either Snc1 or Snc2 (Snc1 or Snc2; plasmids pGADT7-SNC1 and pGADT7-SNC2, respectively), along with a plasmid expressing the Gal4 DNA-binding domain fused to Gcs1 (Gcs1; pGBKT7-GCS1), were transformed into AH109 cells and examined for their ability to grow on medium lacking histidine and containing 3-aminotriazole (3-AT). Cells were plated by serial dilution on selective medium (SC-LT), selective medium lacking histidine (SC-HLT), and the same medium with or without the addition of either 1 mM or 2 mM 3-AT. Negative control (Neg. control) consisted of empty vectors expressing the DNA-binding and TA domains alone. A positive control consisted of an empty vector plus a plasmid (pCL1) expressing full-length Gal4. Cells were grown for 48-72 h at 30°C. (C) HA-Gcs1 coimmunoprecipitates with myc-Snc2. gcs1Δ (MRY4) cells bearing: (1) a multicopy plasmid expressing HA-tagged Gcs1 (pAD54-GCS1) and a single-copy plasmid expressing myc-tagged Snc2 (pHADH-mycSNC2); (2) either expression plasmid (e.g., pAD54-GCS1 or pHADH-mycSNC2) alone along with the appropriate control vector (e.g., pAD54 or pAD11); or (3) both control vectors (pAD54 and pAD11) were grown to log phase, lysed, and processed for coIP with anti-myc antibodies. The immunoprecipitation lanes (IP) and total cell lysate (TCL) shown were detected with anti-Gcs1 antibody (1:2500). (D) The Arf-GAP domain of Gcs1 and amino terminus of Snc2 are dispensable for the Gcs1-Snc2 two-hybrid interaction. Yeast (Y153) was transformed with prey plasmids expressing either Snc2 or a truncated form of Snc2, Snc2^52-115, fused to the TA domain of Gal4 (plasmids pPP381-39 and pSP10C, respectively) and bait plasmids expressing Gcs1 (Gcs1^1-352) or truncated forms of Gcs1 (e.g., Gcs1^1-139; Gcs1^1-226, Gcs1^49-352; Gcs1^117-352; and Gcs1^137-352; plasmids pLM65, pLM61, pLM62, pLM63, and pLM64, respectively) fused to the DNA-binding domain of Gal4. Cells were grown in patches on selective medium, replica plated onto nitrocellulose filters, lysed in liquid nitrogen, and measured for β-galactosidase activity using standard techniques. Shown are a representative filter after β-galactosidase detection (Filter) and a qualitative assessment of β-galactosidase activity (Score).

Snc v-SNAREs and Gcs1 colocalize to late Golgi and endosomal compartments. (A) A subset of internalized Snc1,2 v-SNAREs colocalizes with Gcs1. Single-copy plasmids producing Snc1 or Snc2 tagged with GFP at their amino termini (GFP-Snc1; pRS315-GFP-cSNC1 and GFP-Snc2; pRS315-GFP-SNC2) were transformed into wild-type yeast having a single-copy plasmid expressing Gcs1 tagged at its carboxy terminus with RFP (Gcs1-RFP; YCp50-GCS1-DsRedT4). Cells were visualized by confocal microscopy to show excitation at the appropriate wavelengths (“GFP” and “RFP” windows, respectively). “Merge” represents the merger of the windows. (B) Gcs1 colocalizes with Golgi and early endosomal markers. Wild-type cells bearing an integrated copy of Sec7-RFP (SP1-SEC7RFP), a late Golgi marker, were transformed with single-copy plasmids expressing Gcs1 or Snc2 tagged at the amino terminus with GFP (GFP-Gcs1; pRS315-GFP-GCS1 and GFP-Snc2; pRS315-GFP-SNC2, respectively) and examined for fluorescent marker colocalization. Similarly, cells bearing a single-copy plasmid expressing Gcs1-RFP (YCp50-GCS1DsRedT4) were transformed with plasmids expressing GFP-tagged Tlg2 (GFP-Tlg2; pRS315-GFP-TLG2) or Sed5 (GFP-Sed5; pRS315-GFP-SED5) and examined for colocalization. (C) The Snc1,2 v-SNAREs do not colocalize with Age2. Single-copy plasmids expressing GFP-Snc1 (pRS315-GFP-cSNC1) or GFP-Snc2 (pRS315-GFP-SNC2) and Age2 tagged at the amino terminus with RFP (RFP-Age2; YCp50-DsRedT4-AGE2) were transformed into wild-type cells and visualized by confocal microscopy.

Snc v-SNARE retrieval to the trans-Golgi is defective in gcs1Δ cells. (A) GFP-Snc1 recycling is defective in gcs1Δ cells. Wild-type (SP1), rcy1Δ, gcs1Δ (MRY2), and end4-1 cells expressing GFP-Snc1 from a single-copy plasmid (pRS315-GFP-cSNC1) were grown to midlog phase and processed for confocal fluorescence microscopy. (B) GFP-Snc1 is unable to access the Sec7 compartment (e.g., trans-Golgi) in the absence of Gcs1. Wild-type (SP1) and gcs1Δ (MRY4) cells were transformed with a linearized SEC7-RFP integrating plasmid (YIplac204-T/C-SEC7-dsRED.T4) and correctly integrated SEC7-RFP-expressing cells were transformed with a single copy plasmid expressing GFP-Snc1 (pRS315-GFP-cSNC1), and examined for fluorescence using confocal microscopy. (C) GFP-Snc1 is unable to access the Yif1 compartment (e.g., Golgi) in the absence of Gcs1. Wild-type (SP1) and gcs1Δ (MRY3) cells were transformed with single copy plasmids expressing mRFP-Snc1 (pRS316-mRFP-cSNC1) and GFP-Yif1 (pRS313-GFP-YIF1), and examined for fluorescence using confocal microscopy.

Gcs1 coimmunoprecipitates with Snx4. Wild-type (W303) cells were transformed with plasmids expressing both myc-Snx4 and HA-Gcs1 (pRS426-HA-GCS1 and pAD6-SNX4, respectively), or either myc-Snx4 or HA-Gcs1 alone. Empty vectors (pRS426 or pAD6) were also used as controls. In addition, cells used for a control immunoprecipitation were transformed with plasmids expressing myc-Snc2 and HA-Sso1 (pADH-myc-SNC2 and pRS426-HA-SSO1, respectively) or HA-Sso1 alone. Cells were grown to midlog phase and were processed for immunoprecipitation (see Materials and Methods). Immunoprecipitation (IP) was performed with anti-myc antibodies, whereas Western blotting was performed with both anti-myc and anti-HA antibodies. Blots for both the IP reactions and samples (50 μg) of the total cell lysates (TCL) were probed in parallel.

Deletion of both the SNC1,2 and GCS1 genes results in lethality. (A) gcs1Δ cells are cold-sensitive and are rescued by plasmids expressing GCS1. SP1 wild-type cells and a gcs1Δ disruption strain (MRY1) were transformed with multicopy plasmids expressing SNC1 (pRS426-HA-cSNC1), SNC2 (pRS426-HA-SNC2), GCS1 (pRS426-HA-GCS1), or a single copy plasmid expressing GCS1 fused to RFP (YCp50-GCS1-DsRedT4) were grown to stationary phase (72 h), plated serially on selective medium at various temperatures, and incubated for 2-11 d. Cells bearing empty multicopy (pRS426; vector: 2μ) or single copy (YCp50; vector: CEN) plasmids were used as controls. gcs1Δ cells were grown at 15°C for 11 d; at 18°C for 6 d; and at 30°C for 2 d. Wild-type cells were grown at 15°C for 8 d; at 18°C for 5 d; and at 30°C for 2 d. (B) Combined sncΔ and gcs1Δ null mutations are synthetically lethal. sncΔ (JG8 T15:85) or sncΔ gcs1Δ (MRY5) cells, which both bear plasmid pTGAL-SNC1, were grown to stationary phase (48 h) on galactose-containing medium (which induces expression from the GAL-inducible SNC1 gene). Cells were diluted to 1 OD₆₀₀/ml either in galactose-containing medium or glucose-containing medium (to deplete Snc1) for 24 h, before being serially diluted and plated onto either galactose-containing (Galactose) or glucose-containing (Glucose) solid medium. Cells were grown for 4-11 d on glucose at various temperatures: 15°C, 11 d; 18°C, 8 d; 20°C, 7 d; 30°C, 4 d; 35°C, 4 d; and 37°C, 6 d. Cells were grown for 3-9 d on galactose at various temperatures: 15°C, 9 d; 18°C, 6 d; 20°C, 3 d; 30°C, 3 d; 35°C, 3 d; and 37°C, 4 d.

Snc v-SNAREs bind to Arf1ΔN17-Q71L and coatomer in a Gcs1-dependent manner in vitro and interact genetically with specific COPI subunits. (A) Recombinant Snc2 does not alter Gcs1 Arf-GAP activity in vitro. Recombinant His₆-tagged Gcs1 and His₆-tagged Snc2 (His-Snc2) or His₆-tagged Gcs1 alone (control) were mixed with GTP-bound myristoylated Arf1, and Arf-GAP activity was measured in vitro as described (see Materials and Methods). (B) Recombinant Gcs1 binds to the Snc v-SNAREs in vitro. Purified recombinant Snc1-GST, Snc2-GST, Sec22-GST, or GST alone (5 μg) were incubated with or without recombinant Gcs1 (20 nM) and a GST-pull down assay was performed. Samples subjected to SDS-PAGE and analyzed in immunoblots with anti-Gcs1 antibodies. Ponceau staining of the nitrocellulose filter after gel transfer is shown as a control for the amounts of GST fusion proteins added. Ten percent of added Gcs1 is shown as a control for loading. (C) Recombinant Snc1 binds to Arf1ΔN17-Q71L and purified coatomer in a Gcs1-dependent manner in vitro. Purified recombinant GST-Snc1 or Snc1-GST (5 μg) were incubated with recombinant Gcs1 (20 nM), recombinant Arf1ΔN17-Q71L (7.3 nM), and purified coatomer (40 nM), alone or in combination (see Materials and Methods). Binding was carried out for 1 h at 4°C. Samples were resolved on SDS-PAGE gels and visualized by Fairbanks (Coomassie R) staining. Loadings corresponding to 25% of added Arf1ΔN17-Q71L and 50% of added coatomer are shown in lanes 13 and 14, respectively. (D) GST alone does not bind recombinant Arf1ΔN17-Q71L or purified coatomer. Purified GST was incubated with recombinant Gcs1, recombinant Arf1ΔN17-Q71L, and purified coatomer, alone or in combination, as described above. Binding and detection were performed as under Materials and Methods. (E) SNC1 and SNC2 overexpression inhibits the growth of COPI B subcomplex mutants. sec27-1 and sec33-1 cells were transformed with multicopy plasmids expressing SNC1 (pAD54-cSNC1) or SNC2 (pAD54-SNC2) and either a control vector (pAD54 or pRS426) or a multicopy plasmid expressing GCS1 (pPP329). Cells were grown to midlog phase on selective medium, diluted serially, and plated on solid medium at different temperatures. (F) SNC1 or SNC2 overexpression do not inhibit the growth of a mutant in the COPI F subcomplex. sec21-2 cells were transformed with plasmids expressing SNC1 (pAD54-cSNC1) or SNC2 (pAD54-SNC2) and either a control vector (pAD54) or a multicopy plasmid expressing GCS1 (pPP329). Cells were grown to midlog phase on selective medium, diluted serially, plated on solid medium and incubated at different temperatures for 48 h.

GFP-Snc1 is enriched on the bud plasma membrane in certain COPI mutants and in an ESCRT mutant. Wild-type yeast (W303-1a; WT) and mutants in COPI F (sec21-2), COPI B (sec27-1, sec28Δ, and sec33-1), ESCRT (vps23Δ), RCY1 (rcy1Δ), and END4 (end4-1) were transformed with a single-copy plasmid expressing GFP-Snc1 (pRS315-GFP-cSNC1) and examined by confocal microscopy.

A model for Snc v-SNARE recycling involving Gcs1, Snx4, Rcy1, and COPI. After exocytosis, the Snc1 v-SNARE (designated with red line) undergoes retrieval from the cell surface to the early endosome (EE; light blue fill) and then to the trans-Golgi (blue fill). We propose that in wild-type cells, Gcs1 acts at the level of early endosomes to retrieve Snc1 back to the Golgi. This requires other proteins known to mediate Snc1 recycling to the Golgi, including Snx4 and Rcy1. Thus, Snc1 accumulates at the level of early endosomes in the absence of Gcs1 (gcs1Δ cells). We also propose that a subset of COPI subunits is involved in this step, either directly or indirectly. COPI is known to mediate retrograde Golgi-ER and intra-Golgi transport in yeast and mammals, as well as late endosome (LE)-multivesicular body transport in mammals. Thus COPI, like clathrin, acts as a coat for multiple trafficking pathways. In the absence of certain COPI B subunits (e.g., sec27-1 cells after temperature-shifting or sec28Δ cells), Snc1 is recycled back to the plasma membrane presumably by secretory vesicles derived from endosomal compartments.