Nucleotide Binding by Lhs1p Is Essential for Its Nucleotide Exchange Activity and for Function in Vivo

Abstract

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.

A Nucleotide Exchange Factor Promotes Endoplasmic Reticulum-to-Cytosol Membrane Penetration of the Nonenveloped Virus Simian Virus 40

“…Following reprecipitation of the virus, the samples were subjected to SDS-PAGE and analyzed by silver staining for the biotin-avidin complex and by immunoblotting with anti-VP1 antibodies. dent, consistent with a previous report demonstrating that Grp170's NEF function requires ATP binding (36).…”

“…4C, top panel; compare lanes 1 and 2). This finding indicates that Grp170 but not G41L Grp170 binds to BiP, consistent with a previous report demonstrating that nucleotide binding to Lhs1p is required for efficient Kar2p (yeast BiP homolog) interaction (36). We used a luciferase aggregation assay to further evaluate whether G41L Grp170's ATP-indepen- subjected to the ER-to-cytosol membrane transport assay as described by Inoue and Tsai (14).…”

“…Although Grp170 is also a member of the Hsp70 protein family, it represents an evolutionarily distinct form of canonical Hsp70 proteins that harbor intrinsic ATPase activity. The ATPase activity of yeast Grp170 homolog Lhs1p controls its NEF activity against yeast BiP homolog Kar2p (36). Our data demonstrating that a Grp170 mutant which cannot bind to ATP is NEF defective, cannot stimulate virus ER-to-cytosol transport, and fails to restore SV40 infection in Grp170 knocked-down cells are in line with the notion that Grp170's ATPase activity is essential for its NEF function-this NEF activity is in turn required to release SV40 from BiP prior to viral ER membrane penetration leading to successful infection.…”

“…To this end, we took advantage of a previous study that mutated G28 in the Lhs1p yeast Grp170 homolog to leucine, generating the G28L mutant (36). This mutant cannot bind to ATP due to the presence of the bulky leucine side chain in the nucleotide binding pocket and fails to display any NEF activity.…”

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

“…In a landmark paper, Steel and colleagues discovered that Lhs1 acts as a NEF for Kar2, paving the way for similar revelations about the Hsp110 family (443). Consistent with this role, Lhs1 binds preferentially to the apo-and ADP-bound states of Kar2 and not its ATP-bound state (93). Lhs1 and the Hsp110 Sse1 share key conserved Hsp70-binding residues and employ a similar mechanism to trigger nucleotide exchange on their cognate Hsp70s, Ssa1 and Kar2, respectively (8).…”