Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes - PubMed (original) (raw)
Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes
X Cao et al. J Cell Biol. 2000.
Abstract
Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.
Figures
Figure 1
Subcellular distribution of ER/Golgi proteins by sucrose gradient. Lysed spheroplasts made from CBY409 cells were loaded on a 20–60% sucrose gradient. The gradients were centrifuged at 35,000 rpm in an SW40 rotor for 2.5 h at 4°C. Fractions from the gradient were resolved on SDS-PAGE and immunoblotted for Sec61p (ER marker), Emp47p (Golgi marker), Sec22p, Bet1p, Bos1p (v-SNARE), Sed5p (t-SNARE), and the t-SNARE–associated protein, Sly1p. The relative abundance contained in each fraction was determined by densitometry using NIH image. The GDPase assay measures Ca2+-dependent GDPase activity and serves as a marker for Golgi membranes.
Figure 2
SNARE proteins are incorporated into ER-derived vesicles. COPII-coated vesicles were synthesized from ER membranes and collected by centrifugation. Lanes labeled “Total” are membranes from one-tenth of a total reaction (containing both vesicles and ER). Lanes labeled “+Recon” are vesicles produced under conditions of reconstituted vesicle formation by addition of COPII proteins. Lanes labeled “−Recon” are those produced in the absence of COPII proteins. Samples were resolved on 12.5% SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies specific for indicated proteins.
Figure 3
The v-SNAREs Bet1p and Bos1p are functionally required on ER-derived vesicles. COPII vesicles, which were synthesized in vitro from wild-type or mutant ER membranes at 20°C, were incubated in a second stage with wild-type or mutant Golgi membranes at 23 or 29°C. (A) Vesicle fusion with membrane components from a bet1-1 strain or (B) a bos1-1 strain. Reactions contained an ATP regeneration system alone (Mem. + Ves., open bars) or an ATP regeneration system with Uso1p and LMA1 (Mem. + Ves. + Fusion Factors, black bars). The percent fusion was quantified after precipitation of the outer chain–modified forms of [35S]gp-αF.
Figure 4
The t-SNARE Sed5p is functionally required on Golgi membranes. COPII vesicles synthesized in vitro from wild-type or sed5-1 ER membranes at 20°C were incubated in a second stage with wild-type or sed5-1 Golgi membranes at 23 or 29°C. Reactions contained an ATP regeneration system alone (Mem. + Ves., open bars) or an ATP regeneration system with Uso1p and LMA1 (Mem. + Ves. + Fusion Factors, black bars). The percent fusion represents the amount of the outer chain–modified forms of [35S]gp-αF.
Figure 5
The v-SNARE Bet1p is required on ER-derived vesicles, and the t-SNARE Sed5p is required on Golgi membranes. Vesicles synthesized from a bet1-1 strain and Golgi membranes from a sed5-1 strain or vice versa were incubated in a second stage at 23 or 29°C. Reactions contained an ATP regeneration system alone (Mem. + Ves., open bars) or an ATP regeneration system with Uso1p and LMA1 (Mem. + Ves. + Fusion Factors, black bars). The percent fusion represents the amount of the outer chain–modified forms of [35S]gp-αF.
Figure 6
Sly1p function is required on Golgi membranes. COPII vesicles synthesized in vitro from wild-type or sly1-ts ER membranes at 20°C were incubated in a second stage with wild-type or sly1-ts Golgi membranes at 23 or 29°C. Reactions contained an ATP regeneration system alone (Mem. + Ves., open bars) or an ATP regeneration system with Uso1p and LMA1 (Mem. + Ves. + Fusion Factors, black bars). The percent fusion represents the amount of outer chain–modified forms of [35S]gp-αF.
Figure 7
The small GTPase Ypt1p is functionally required on Golgi membranes. COPII vesicles, which were synthesized in vitro from wild-type or ypt1-3 membranes at 20°C, were incubated in a second stage with wild-type or ypt1-3 Golgi membranes at 23 or 29°C. Reactions contained an ATP regeneration system alone (Mem. + Ves., open bars) or an ATP regeneration system with Uso1p and LMA1 (Mem. + Ves. + Fusion Factors, black bars). The percent fusion represents the amount of the outer chain–modified forms of [35S]gp-αF.
Figure 9
Ypt1-TM2p functions on Golgi membranes. COPII vesicles synthesized in vitro from ypt1-3 membranes were incubated in a second stage with wild-type or Ypt1-TM2p Golgi membranes. Reactions contained an ATP regeneration system alone (No addition, open bars) or supplemented with Uso1p and LMA1 (Fusion Factors, black bars) or Uso1p and LMA1 and 50 μg/ml Gdi1p (Fusion Factors + GDI, hatched bars). The percent fusion represents the amount of outer chain–modified forms of [35S]gp-αF.
Figure 8
Gdi1p extracts Ypt1p but not Ypt1-TM2p. Wild-type (WT) or Ypt1-TM2p (TM2) semi-intact cells were incubated with Gdi1p (50 μg/ml) for 20 min at 25°C. Total reactions (T) were centrifuged at 100,000 g to generate the membrane pellet (P) and supernatant (S) fractions. Fractions were resolved on 12.5% polyacrylamide gels, and immunoblot analyses were performed with specific antibodies to measure the content of Sec23p (a peripheral membrane protein control), Sec61p (control for integral membrane protein), and the lipid-anchored or membrane-anchored form of Ypt1p.
Comment in
- Reversal of fortune. Do Rab GTPases act on the target membrane?
Brennwald P. Brennwald P. J Cell Biol. 2000 Apr 3;149(1):1-4. doi: 10.1083/jcb.149.1.1. J Cell Biol. 2000. PMID: 10747079 Free PMC article. No abstract available.
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