The Gcs1 Arf-GAP mediates Snc1,2 v-SNARE retrieval to the Golgi in yeast.
Gcs1 is an Arf GTPase-activating protein (Arf-GAP) that mediates Golgi-ER and post-Golgi vesicle transport in yeast. Here we show that the Snc1,2 v-SNAREs, which mediate endocytosis and exocytosis, interact physically and genetically with Gcs1. Moreover, Gcs1 and the Snc v-SNAREs colocalize to subcellular structures that correspond to the trans-Golgi and endosomal compartments. Studies performed in vitro demonstrate that the Snc-Gcs1 interaction results in the efficient binding of recombinant Arf1Delta17N-Q71L to the v-SNARE and the recruitment of purified coatomer. In contrast, the presence of Snc had no effect on Gcs1 Arf-GAP activity in vitro, suggesting that v-SNARE binding does not attenuate Arf1 function. Disruption of both the SNC and GCS1 genes results in synthetic lethality, whereas overexpression of either SNC gene inhibits the growth of a distinct subset of COPI mutants. We show that GFP-Snc1 recycling to the trans-Golgi is impaired in gcs1Delta cells and these COPI mutants. Together, these results suggest that Gcs1 facilitates the incorporation of the Snc v-SNAREs into COPI recycling vesicles and subsequent endosome-Golgi sorting in yeast.
Robinson
Micah
M
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Poon
Pak Phi
PP
Schindler
Christina
C
Murray
Lois E
LE
Kama
Rachel
R
Gabriely
Galina
G
Singer
Richard A
RA
Spang
Anne
A
Johnston
Gerald C
GC
Gerst
Jeffrey E
JE
D022181:COP-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D005804:Genes, Lethal; D006056:Golgi Apparatus / Q000378:metabolism*; D047468:Immunoprecipitation; D025941:Protein Interaction Mapping; D021381:Protein Transport; D050683:R-SNARE Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000737:chemistry / Q000378:metabolism*
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D050683:R-SNARE Proteins