Localization, dynamics, and protein interactions reveal distinct roles for ER and Golgi SNAREs.
ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.
Hay
J C
JC
Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5428, USA.
Klumperman
J
J
Oorschot
V
V
Steegmaier
M
M
Kuo
C S
CS
Scheller
R H
RH
D000818:Animals; D019556:COS Cells; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D016716:PC12 Cells; D011233:Precipitin Tests; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D011817:Rabbits; D051381:Rats; D050600:SNARE Proteins; D013347:Subcellular Fractions; D013696:Temperature; D014407:Tumor Cells, Cultured; D033921:Vesicular Transport Proteins
C098783:BET1 protein, rat; C491344:GOSR2 protein, human; C105309:Gosr2 protein, mouse; C491343:Gosr2 protein, rat; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C484373:SEC22A protein, human; D050600:SNARE Proteins; C098784:Sec22a protein, rat; D033921:Vesicular Transport Proteins