Graham Warren

Implications of the SNARE hypothesis for intracellular membrane topology and dynamics

The SNARE hypothesis provides a mechanism for the specific docking and fusion of transport vesicles with their target membranes. A simple extension of the hypothesis can explain many cellular processes, including the stacking of Golgi cisternae, retrograde transport and homotypic fusion; it can also explain the morphology of intracellular membranes and their dynamics during mitosis.

The Road Taken: Past and Future Foundations of Membrane Traffic

We thank the members of the newly formed Mellman/Warren lab, as well as many of our colleagues both at Yale and elsewhere, for their opinions, criticism, and patience during the writing of this review. In particular, we thank Jim Jamieson at Yale not only for having performed one of the most important founding experiments in membrane traffic, but also for having kindly provided the data that we used for Figure 1Figure 1.We also thank the National Institutes of Health and the Ludwig Institute for Cancer Research for their generous support and continuing encouragement of our work.This review is dedicated to the memory of Thomas E. Kreis, friend and colleague, who to our everlasting regret did not survive to witness the start of the next millennium.

A complex of mammalian Ufd1 and Npl4 links the AAA‐ATPase, p97, to ubiquitin and nuclear transport pathways

The AAA‐ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin‐dependent protein degradation. Binding of the p47 complex directs p97 to act in the post‐mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin‐dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by p97, the most likely being ubiquitin‐dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways.

p47 is a cofactor for p97-mediated membrane fusion

At least two distinct ATPases, NSF and p97, are known to be involved in the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of membrane compartments. The NSF-mediated fusion pathway is the best characterized, many of the components having been identified and their functions analysed. In contrast, none of the accessory proteins for the p97-mediated fusion pathway has been identified. Now we have identified the first such component, a protein of relative molecular mass 47,000 (p47), which forms a tight, stoichiometric complex with cytosolic p97 (one trimer of p47 per hexamer of p97). It is essential for the p97-mediated regrowth of Golgi cisternae from mitotic Golgi fragments, a process restricted to animal cells. As a homologue of p47 exists in budding yeast, this indicates that it might also be involved in other membrane fusion reactions catalysed by p97, such as karyogamy.

Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells

The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B–dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B–dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein in a mitotically regulated manner

The docking of transport vesicles with their target membrane is thought to be mediated by p115. We show here that GM130, a cis-Golgi matrix protein, interacts specifically with p115 and so could provide a membrane docking site. Deletion analysis showed that the N-terminus binds to p115, whereas the C-terminus binds to Golgi membranes. Mitotic phosphorylation of GM130 or a peptide derived from the N-terminus prevented binding to p115. The peptide also inhibited the NSF- but not the p97-dependent reassembly of Golgi cisternae from mitotic fragments, unless it was mitotically phosphorylated. Together, these data provide a molecular explanation for the COPI-mediated fragmentation of the Golgi apparatus at the onset of mitosis.

GRASP65, A PROTEIN INVOLVED IN THE STACKING OF GOLGI CISTERNAE

NEM prevents mitotic reassembly of Golgi cisternae into stacked structures. The major target of NEM is a 65 kDa protein conserved from yeast to mammals. Antibodies to this protein and a recombinant form of it block cisternal stacking in a cell-free system, justifying its designation as a Golgi ReAssembly Stacking Protein (GRASP65). One of the two minor targets of NEM is GM130, previously implicated in the docking of transport vesicles and mitotic fragmentation of the Golgi stack. GRASP65 is complexed with GM130 and is tightly bound to Golgi membranes, even under mitotic conditions when both are heavily phosphorylated. These results link vesicle docking, stacking of Golgi cisternae, and the disruption of both of these interactions during mitosis.

Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly.

Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97–Ufd1–Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97–p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.

Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1–Npl4

The multiple functions of the p97/Cdc48p ATPase can be explained largely by adaptors that link its activity to different cellular pathways, but how these adaptors recognize different substrates is unclear. Here we present evidence that the mammalian adaptors, p47 and Ufd1–Npl4, both bind ubiquitin conjugates directly and so link p97 to ubiquitylated substrates. In the case of Ufd1–Npl4, which is involved in endoplasmic reticulum (ER)‐associated degradation and nuclear envelope reassembly, binding to ubiquitin is mediated through a putative zinc finger in Npl4. This novel domain (NZF) is conserved in metazoa and is both present and functional in other proteins. In the case of p47, which is involved in the reassembly of the ER, the nuclear envelope and the Golgi apparatus, binding is mediated by a UBA domain. Unlike Ufd1–Npl4, it binds ubiquitin only when complexed with p97, and binds mono‐ rather than polyubiquitin conjugates. The UBA domain is required for the function of p47 in mitotic Golgi reassembly. Together, these data suggest that ubiquitin recognition is a common feature of p97‐mediated reactions.

Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol

A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer. Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness in vivo because measurements in vitro have shown that cholesterol can increase the thickness of simple phospholipid/cholesterol bilayers. Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol. The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 ± 0.4 Å, 39.5 ± 0.4 Å, 35.6 ± 0.6 Å, and 42.5 ± 0.3 Å, respectively. After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 Å. Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness. Protein-depleted membranes demonstrated changes in thickness of up to 5 Å, suggesting that (i) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (ii) proteins and lipids are not naturally hydrophobically matched in some biological membranes. A marked effect of membrane proteins on the thickness of Escherichia coli cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings.