Saccharomyces cerevisiaeRot1 Is an Essential Molecular Chaperone in the Endoplasmic Reticulum

Abstract

Molecular chaperones prevent aggregation of denatured proteins in vitro and are thought to support folding of diverse proteins in vivo. Chaperones may have some selectivity for their substrate proteins, but knowledge of particular in vivo substrates is still poor. We here show that yeast Rot1, an essential, type-I ER membrane protein functions as a chaperone. Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation. In vivo, the rot1-2 mutation caused accelerated degradation of five proteins in the secretory pathway via ER-associated degradation, resulting in a decrease in their cellular levels. Furthermore, we demonstrate a physical and probably transient interaction of Rot1 with four of these proteins. Collectively, these results indicate that Rot1 functions as a chaperone in vivo supporting the folding of those proteins. Their folding also requires BiP, and one of these proteins was simultaneously associated with both Rot1 and BiP, suggesting that they can cooperate to facilitate protein folding. The Rot1-dependent proteins include a soluble, type I and II, and polytopic membrane proteins, and they do not share structural similarities. In addition, their dependency on Rot1 appeared different. We therefore propose that Rot1 is a general chaperone with some substrate specificity.

Kre6 Protein Essential for Yeast Cell Wall β-1,6-Glucan Synthesis Accumulates at Sites of Polarized Growth

“…2). The calculated molecular weight of Kre6 is 80,122, but the detected Kre6 is significantly larger than the expected size, probably because of N-glycosylation and phosphorylation (3,12). We concluded a specific antiKre6 antiserum was obtained that specifically recognizes Kre6 but not Skn1.…”

“…The loss of Rot1 in the ER results in a severe growth defect and reduction of the cell wall ␤-1,6-glucan content. Takeuchi et al (12) showed that Rot1 functions as the molecular chaperon of Kre6 and Kre5 and is engaged in the folding and stabilization of these proteins. They suggested that the reduction of the ␤-1,6-glucan content in the ⌬rot1 cells may be a secondary effect through the defect of Kre6 and Kre5 (12).…”

“…The double-ring ER profiles were clearly observed, and its interaction with ERresident essential protein Keg1 supported this localization of Kre6 (9). Takeuchi et al (12) showed that an ER chaperon Rot1 and ubiquitin ligase Ubc7 affects the stability of Kre6 and suggested that Kre6 recycles between the ER and Golgi based on the results of sucrose gradient cell fractionation.…”

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

“…The Glc 3 Man 9 GlcNAc 2 glycan, which comprises the N-linked core, is rapidly processed by glucosidase I (Gls1/ Cwh41) and glucosidase II (Gls2/Rot2) enzymes to remove the three terminal glucose residues and generate Man 9-GlcNAc 2 . Molecular chaperones collaborate in protein folding during these glucose-trimming events and Rot1 alone has been shown to possess a general chaperone activity (Takeuchi et al 2008). In many cell types, a calnexindependent folding cycle operates to iteratively fold and monitor polypeptide status through the coordinated activities of glucosidase I, glucosidase II, UDP-glucose;glycoprotein glucosyltransferase (UGGT), and calnexin (Cne1).…”

Impact of Yeast Glycosylation Pathway on Cell Integrity and Morphology

“…In addition, the sec 59-1 phenotype could be rescued by overexpression of the ROT1 gene, encoding the endoplasmic reticulum Rot1 protein. The latter is implicated in cell wall biogenesis and acts as a chaperone for misfolded proteins [36,37]. Recently, we have shown also that Rot1 interacts with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation.…”