Identification of Essential Residues in the Type II Hsp40 Sis1 That Function in Polypeptide Binding

Abstract

Sis1 is an essential yeast Type II Hsp40 protein that assists cytosolic Hsp70 Ssa1 in the facilitation of processes that include translation initiation, the prevention of protein aggregation, and proteasomal protein degradation. An essential function of Sis1 and other Hsp40 proteins is the binding and delivery of non-native polypeptides to Hsp70. How Hsp40s function as molecular chaperones is unknown. The crystal structure of a Sis1 fragment that retains peptide-binding activity suggests that Type II Hsp40s utilize hydrophobic residues located in a solvent-exposed patch on carboxyl-terminal domain I to bind non-native polypeptides. To test this model, amino acid residues Val-184, Leu-186, Lys-199, Phe-201, Ile-203, and Phe-251, which form a depression in carboxyl-terminal domain I, were mutated, and the ability of Sis1 mutants to support cell viability and function as molecular chaperones was examined. We report that Lys-199, Phe-201, and Phe-251 are essential for cell viability and required for Sis1 polypeptide binding activity. Sis1 I203T could support normal cell growth, but when purified it exhibited severe defects in chaperone function. These data identify essential residues in Sis1 that function in polypeptide binding and help define the nature of the polypeptide-binding site in Type II Hsp40 proteins.

Functions of Yeast Hsp40 Chaperone Sis1p Dispensable for Prion Propagation but Important for Prion Curing and Protection From Prion Toxicity

“…The adjacent glycinephenylalanine (GF)-rich region is important for specifying Sis1p function and for propagation of the [PIN 1 ]/ [RNQ 1 ] prion (Yan and Craig 1999;Sondheimer et al 2001), and the glycine-methionine (GM)-rich region contributes to specificity of substrate interaction (Fan et al 2004). The C-terminal domain (CTDI and CTDII) and residue K199 are important for substrate binding (Lee et al 2002) and the 14 C-terminal residues comprise a motif (DD) that promotes dimerization (Sha et al 2000). Protein alterations are indicated on the left.…”

Functional Analysis of CbpA, a DnaJ Homolog and Nucleoid-associated DNA-binding Protein

“…The location of the peptide binding site of a type II DnaJ homolog, Ydj1, is known from crystallographic studies (7). In addition, the peptide binding site in yeast Sis1 has been identified by genetic and biochemical studies (10,27 (Fig. 2, B and C).…”

Role of DnaJ G/F-rich Domain in Conformational Recognition and Binding of Protein Substrates*

“…The second essential function of Hsp40s is the binding and delivery of substrate proteins to Hsp70 (18,19), a process not well understood. Type I Hsp40s can act as independent chaperones and bind and suppress aggregation of non-native polypeptides (20,21), while type II proteins have to cooperate with Hsp70s to prevent aggregation (22). The crystal structure of Ydj1p and Sis1p reveals a hydrophobic pocket in the C-terminal domain that can accommodate short peptides (23,24).…”