Hsp70s are a ubiquitous family of molecular chaperones involved in many cellular processes. Two Hsp70s, Lhs1p and Kar2p, are required for protein biogenesis in the yeast endoplasmic reticulum. Here, we found that Lhs1p and Kar2p specifically interacted to couple, and coordinately regulate, their respective activities. Lhs1p stimulated Kar2p by providing a specific nucleotide exchange activity, whereas Kar2p reciprocally activated the Lhs1p adenosine triphosphatase (ATPase). The two ATPase activities are coupled, and their coordinated regulation is essential for normal function in vivo.

Protein translocation and folding in the endoplasmic reticulum of Saccharomyces cerevisiae involves two distinct Hsp70 chaperones, Lhs1p and Kar2p. Both proteins have the characteristic domain structure of the Hsp70 family consisting of a conserved N-terminal nucleotide binding domain and a C-terminal substrate binding domain. Kar2p is a canonical Hsp70 whose substrate binding activity is regulated by cochaperones that promote either ATP hydrolysis or nucleotide exchange. Lhs1p is a member of the Grp170/Lhs1p subfamily of Hsp70s and was previously shown to function as a nucleotide exchange factor (NEF) for Kar2p. Here we show that in addition to this NEF activity, Lhs1p can function as a holdase that prevents protein aggregation in vitro. Analysis of the nucleotide requirement of these functions demonstrates that nucleotide binding to Lhs1p stimulates the interaction with Kar2p and is essential for NEF activity. In contrast, Lhs1p holdase activity is nucleotide-independent and unaffected by mutations that interfere with ATP binding and NEF activity. In vivo, these mutants show severe protein translocation defects and are unable to support growth despite the presence of a second Kar2p-specific NEF, Sil1p. Thus, Lhs1p-dependent nucleotide exchange activity is vital for ER protein biogenesis in vivo.

Tail-anchored or C-terminally-anchored proteins play many essential roles in eukaryotic cells. However, targeting and insertion of this class of membrane protein has remained elusive. In this study, we reconstitute insertion of tail-anchored proteins into microsomes derived from Saccharomyces cerevisiae. Using this approach, we are able to genetically manipulate the composition of the microsomes in order to address the question of which components of the endoplasmic reticulum (ER) are required for this process. We show that tail-anchored protein insertion is not dependent on the classical SEC translocation machinery but rather occurs via an ATP-dependent pathway involving at least one novel membrane protein factor. We further demonstrate that the specificity of this pathway is conserved between yeast and mammals.

The SEC18 gene product is 48% identical to mammalian NSF (N-ethylmaleimide-sensitive fusion protein), and both proteins encode cytoplasmic ATPases which are essential for membrane traffic in yeast and mammalian cells, respectively. A wealth of biochemical analysis has led to the description of a model for the action of NSF; through its interaction with SNAPs (soluble NSF attachment proteins), NSF can associate with SNAP receptors (SNAREs) on intracellular membranes, forming 20S complexes. SNAPs then stimulate the intrinsic ATPase activity of NSF, leading to the disassembly of the 20S complex, which is essential for subsequent membrane fusion. Although this model is based almost entirely on in vitro studies of the original clones of NSF and alpha-SNAP, it is nevertheless widely assumed that this mechanism of membrane fusion is conserved in all eukaryotic cells. If so, the crucial biochemical properties of NSF and SNAPs should be shared by their yeast homologues, Sec18p and Sec17p. Using purified recombinant proteins, we report here that Sec18p can specifically interact not only with Sec17p but also with its mammalian homologue, alpha-SNAP. This interaction leads to a stimulation of Sec18p D1 domain ATPase activity, with kinetics similar to those of alpha-SNAP stimulation of NSF, although differences in temperature and N-ethylmaleimide sensitivity were observed between NSF and Sec18p. Furthermore, Sec18p can interact with synaptic SNARE proteins and can synergize with alpha-SNAP to stimulate regulated exocytosis in mammalian cells. We conclude that the mechanistic properties of NSF and SNAPs are shared by Sec18p and Sec17p, thus demonstrating that the biochemistry of membrane fusion is conserved from yeast to mammals.

N-Ethylmaleimide-sensitive fusion protein (NSF) is required for most intracellular membrane fusion events. NSF is recruited to membranes by soluble NSF attachment proteins (SNAPs) and membrane-resident SNAP receptor (SNARE) proteins. The 20 S complex of NSF/SNAPs/SNAREs disassembles when NSF hydrolyses ATP, and this disassembly event is believed to be essential for membrane fusion. SNAPs stimulate NSF ATPase activity, but it is not known which of NSF's two ATPase domains (D1 or D2) is affected. Using recombinant mutant NSFs defective in ATP hydrolysis in one domain only, we found that SNAPs stimulate NSF ATPase activity by a selective action on the D1 domain, yet had no effect on the D2 domain. Since the D1 domain of NSF is implicated in 20 S complex disassembly, this supports the idea that SNAP stimulation of NSF ATPase activity is required for membrane fusion.z 1998 Federation of European Biochemical Societies.