Distinct SNARE complexes mediating membrane fusion in Golgi transport based on combinatorial specificity.
Syntaxin-5 (Sed5) is the only syntaxin needed for transport into and across the yeast Golgi, raising the question of how a single syntaxin species could mediate vesicle transport in both the anterograde and the retrograde direction within the stack. Sed5 is known to combine with two light chains (Bos1 and Sec22) to form the t-SNARE needed to receive vesicles from the endoplasmic reticulum. However, the yeast Golgi contains several other potential light chains with which Sed5 could potentially combine to form other t-SNAREs. To explore the degree of specificity in the choice of light chains by a t-SNARE, we undertook a comprehensive examination of the capacity of all 21 Sed5-based t-SNAREs that theoretically could assemble in the yeast Golgi to fuse with each of the 7 potential v-SNAREs also present in this organelle. Only one additional of these 147 combinations was fusogenic. This functional proteomic strategy thereby revealed a previously uncharacterized t-SNARE in which Sed5 is the heavy chain and Gos1 and Ykt6 are the light chains, and whose unique cognate v-SNARE is Sft1. Immunoprecipitation experiments confirmed the existence of this complex in vivo. Fusion mediated by this second Golgi SNAREpin is topologically restricted, and existing genetic and morphologic evidence implies that it is used for transport across the Golgi stack. From this study, together with the previous functional proteomic analyses which have tested 275 distinct quaternary SNARE combinations, it follows that the fusion potential and transport pathways of the yeast cell can be read out from its genome sequence according to the SNARE hypothesis with a predictive accuracy of about 99.6%.
Parlati
Francesco
F
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 251, New York, NY 10021, USA.
Varlamov
Oleg
O
Paz
Keren
K
McNew
James A
JA
Hurtado
David
D
Söllner
Thomas H
TH
Rothman
James E
JE
D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; C490973:GOS1 protein, S cerevisiae; D008565:Membrane Proteins; D010455:Peptides; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C094380:SFT1 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C077554:Sed5 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C107127:YKT6 protein, S cerevisiae; C491481:Ykt6 protein, rat; D005982:Glutathione Transferase