Cer1p Functions as a Molecular Chaperone in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Abstract

Cer1p/Lhs1p/Ssi1p is a novel Hsp70-related protein that is important for the translocation of a subset of proteins into the yeast Saccharomyces cerevisiae endoplasmic reticulum. Cer1p has very limited amino acid identity to the hsp70 chaperone family in the N-terminal ATPase domain but lacks homology to the highly conserved hsp70 peptide binding domain. The role of Cer1p in protein folding and translocation was assessed. Deletion of CER1 slowed the folding of reduced pro-carboxypeptidase Y (pro-CPY) approximately twofold in yeast. In wild-type yeast under reducing conditions, pro-CPY can be found in a complex with Cer1p, while partially purified Cer1p is able to bind directly to peptides. Together, this suggests that Cer1p has a chaperoning activity required for proper refolding of denatured pro-CPY which is mediated by direct interaction with the unfolded polypeptide. Cer1p peptide binding and oligomerization could be disrupted by addition of ATP, confirming that Cer1p possesses a functional ATP binding site, much like Kar2p and other members of the hsp70 family. Interestingly, replacing the signal sequence of a CER1-dependent protein with that of a CER1-independent protein did not relieve the requirement of CER1 for import. This result suggests that an interaction with the mature portion of the protein also is important for the translocation role of Cer1p. The CER1 RNA levels increase at lower temperatures. In addition, the effects of deletion on folding and translocation are more severe at lower temperatures. Therefore, these results suggest that Cer1p provides an additional chaperoning activity in processes known to require Kar2p. However, there appears to be a greater requirement for Cer1p chaperone activity at lower temperatures.

LHS1 and SIL1 provide a lumenal function that is essential for protein translocation into the endoplasmic reticulum

“…Current evidence indicates that Kar2p/BiP is sufficient to gate the translocon during initiation of translocation (Hamman et al ., 1998). However, the Lhs1 protein (Lhs1p) has been shown to bind to peptides in an ATP‐dependent manner similar to that demonstrated for BiP (Hamilton et al ., 1999). We therefore propose that Lhs1p binds to translocating polypeptides, thus contributing to the driving force for post‐translational translocation in a manner similar to that previously suggested for Kar2p.…”

Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems

“…The reason for, or mechanism of, this induction is not currently known, although the protein product of Bfr2, Bfr2p, involved in protein trafficking to the Golgi, is thought to be essential for mass growth and/or cell proliferation in S. cerevisiae [59]. Similarly, the mRNA levels of Cer1p, an Hsp70-related protein involved in the translocation of a subset of proteins into the endoplasmic reticulum in S. cerevisiae, increases upon cold shock, and it has been suggested that there is enhanced demand for the chaperone activity of Cer1p at lower temperatures [60].…”

The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum

“…LHS1/CER1/SSI1 encodes a non-essential Hsp70 of the ER. Δlhs1 cells are viable at all temperatures but exhibit a partial translocation block of some ER proteins and defects in protein folding [17][18][19]39]. We generated a double disrupted Δlhs1Δerj5 strain and found that the strain was viable and, different than the kar2-159Δerj5 mutant, did not show any detectable growth defect at any temperature tested or in presence of agents that disturb the folding capacity of the ER.…”

“…JEM1 has also been found to function in karyogamy in conjunction with KAR2 [15,16]. Loss of the nonessential Lhs1p causes a translocation defect of a subset of proteins into the ER and reduced ability to fold proteins in the ER lumen, suggesting a partial overlap of functions between the two Hsp70s [17][18][19]. Recent studies demonstrated that Kar2p and Lhs1p interact with each other to couple their respective activities, and that the two Hsp70s do not share a common complement of cochaperones [20].…”