The formation of golgi stacks from vesiculated golgi membranes requires two distinct fusion events

Abstract

We have reconstituted the fusion and assembly of vesiculated Golgi membranes (VGMs) into functionally active stacks of cisternae. A kinetic analysis of this assembly process revealed that highly dispersed VGMs of 60-90 nm diameter first fuse to form larger vesicles of 200-300 nm diameter that are clustered together. These vesicles then fuse to form tubular elements and short cisternae, which finally assemble into stacks of cisternae. We now provide evidence that the sequential stack formation from VGMs reflects two distinct fusion processes: the first event is N-ethyl-maleimide (NEM)-sensitive factor (NSF) dependent, and the second fusion event requires an NSF-like NEM-sensitive ATPase called p97. Interestingly, while the earliest steps in stack formation share some similarities with events catalyzing fusion of transport vesicles to its target membrane, neither GTP gamma S nor Rab-GDI, inhibitors of vesicular protein traffic, inhibit stack formation.

SPAF, a new AAA-protein specific to early spermatogenesis and malignant conversion

“…Members of this gene family are present in prokaryocytes and eukaryotes, including bacteria (Confalonieri et al, 1994), yeast , plants (Feiler et al, 1995), and mammals (Egerton et al, 1992). They function in a variety of cellular processes, including vesicle-mediated transport (Eakle et al, 1988;Wilson et al, 1989;Babst et al, 1997), cell cycle regulation Clark-Maguire and Mains, 1994), peroxisome biogenesis , transcriptional regulation (Nelbock et al, 1990), and membrane fusion (Rabouille et al, 1995;Acharya et al, 1995). In S. cerevisiae alone, 22 di erent proteins with AAA-protein motifs have been identi®ed, suggesting that there are at least as many biological functions associated with this family (Beyer, 1997).…”

“…PAS is another member of the subfamily involved in membrane fusion events, whose mutant is incapable of forming peroxisomes from smaller precursors . Recently, two other kinds of membrane fusion events, ER fusion and Golgi cisternae formation, have been associated with AAA-proteins CDC48 (Latterich et al, 1995) and p97ATP-ase (Rabouille et al, 1995;Acharya et al, 1995), respectively. To date, however, none of the members in this subfamily have been associated with the membrane fusion events of mitochondria, one of the most abundant membrane-containing cellular organelles.…”

Biogenesis of Golgi Stacks in Imaginal Discs ofDrosophila melanogaster

“…Bar, 100 nm. cisternae in vitro, NSF (and its cofactor ␣SNAP), and p97 (and its cofactor p47) (Acharya et al, 1995;Rabouille et al, 1995a;Kondo et al, 1997). We tested the involvement of dNSF1 (one of the two Drosophila homologs of mammalian NSF) in the observed Golgi stack biogenesis with the use of larvae from comt 17 homozygote stocks (gifts from Dr. B. Ganetsky).…”

“…The in vitro assay measured the rebuilding of the Golgi apparatus from mitotic Golgi fragments (Rabouille et al, 1995b). The semi-intact cell system visualized the rebuilding of the Golgi complex from illimaquinone-generated Golgi fragments (Acharya et al, 1995). These assays have allowed the identification of several proteins involved in this rebuilding process, such as NEM sensitive factor (NSF) and its cofactor ␣ soluble NSF attachment protein (a-SNAP) (Acharya et al, 1995;Rabouille et al, 1995a;Mü ller et al, 1999), p97 (Acharya et al, 1995;Rabouille et al, 1995a), and its cofactor p47 (Kondo et al, 1997).…”

The Evolution of the Conserved ATPase Domain (CAD): Reconstructing the History of an Ancient Protein Module

“…NSF/ Sec18p is also involved in the reformation of Golgi stacks after mitotic fragmentation. This process further requires the activity of another member of this group of AAA proteins, VCP/p97 (the homologue of yeast Cdc48), at a later stage of the process (Acharya et al 1995;Rabouille et al 1995). VCP/p97 also interacts with the vesicle-coating protein clathrin (Pleasure et al 1993) and in human T-cells is tyrosine-phosphorylated in response to proliferation signals (Schulte et al 1994).…”