Asymmetric Requirements for a Rab Gtpase and Snare Proteins in Fusion of Copii Vesicles with Acceptor Membranes

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.

Erv26p Directs Pro-Alkaline Phosphatase into Endoplasmic Reticulum–derived Coat Protein Complex II Transport Vesicles

“…Under steady state conditions, ϳ60% of the Erv26p cofractionated with the Golgi marker protein Och1p and ϳ40% with the ER marker Sec61p. This subcellular distribution pattern was similar to other COPII vesicles proteins including Emp24p, Erv46p and ER/Golgi SNARE proteins (Schimmoller et al, 1995;Otte et al, 2001;Cao and Barlowe, 2000).…”

“…Under steady state conditions, ϳ60% of the Erv26p cofractionated with the Golgi marker protein Och1p and ϳ40% with the ER marker Sec61p. This subcellular distribution pattern was similar to other COPII vesicles proteins including Emp24p, Erv46p and ER/Golgi SNARE proteins (Schimmoller et al, 1995;Otte et al, 2001;Cao and Barlowe, 2000).To test whether Erv26p dynamically cycles between the ER and Golgi compartments in vivo, we monitored the fate of this protein in a sec12 mutant strain. The sec12 mutation blocks COPII budding when shifted to a restrictive temperature (Kaiser and Schekman, 1990); therefore, if Erv26p actively cycles between the ER and Golgi, we would expect it to accumulate in the ER under a sec12 block.…”

Identification of rab20 as a Potential Regulator of Connexin43 Trafficking

“…rab family proteins are small-molecular-weight GTPases, which associate with distinct intracellular membrane compartments and regulate membrane traffic (Zerial and McBride 2001). The best characterized rab function is regulation of membrane fusion by cycling between an active, membrane associated, GTP-bound state and an inactive, GDP-bound state (Barbieri et al 1998;Cao and Barlowe 2000;Grindstaff et al 1998). Once at the membrane, rabs orchestrate the assembly of membrane fusion machinery (Carr et al 1999).…”

The Yip1p·Yif1p Complex Is Required for the Fusion Competence of Endoplasmic Reticulum-derived Vesicles

“…Lysates were prepared from SFNY1105 as described above and fractionated on a gradient previously used to separate the ER and Golgi where Ypt1p is normally found (22,29). The SNARE Bos1p marks the Golgi (Fig.…”