Causal Links Between Protein Folding in the ER and Events Along the Secretory Pathway

Takeuchi, Masato; Kohno, Kenji

doi:10.4161/auto.3089

Cited by 3 publications

(2 citation statements)

References 25 publications

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“…, encoding an essential ER-localized membrane protein, was initially reported as a gene required for ß-1,6-glucan synthesis (Bickle et al 1998;Machi et al 2004). Mutations of ROT1 cause pleiotropic defects in cell wall synthesis, cytoskeleton dynamics, or in lysis of autophagic bodies (Takeuchi et al 2006a;Takeuchi et al 2006b). More recently it was demonstrated that Rot1 can function as a general chaperone exhibiting anti-aggregation activity in vitro, and in vivo the rot1-2 mutation caused an accelerated degradation of several proteins of the secretory pathway via ER-associated degradation (Takeuchi et al 2008).…”

Section: Rot1mentioning

confidence: 99%

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

2012

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Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3∆ cells was found to be decreased to 45% compared to wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an underglycosylation pattern. By affinity chromatography a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation process, as glycosylation of distinct glycoproteins of this type were affected as well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation.

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Section: Rot1mentioning

confidence: 99%

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

2012

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“…In eukaryotic cells, intracellular degradation of mutated proteins was known to depend on two main proteolytic pathways: endoplasmic reticulum (ER) associated degradation through proteasomes or autophagy through lysosomes . Most secretory proteins enter the ER to undergo posttranslational modifications and folding prior to their transit to the Golgi and subsequently to the cell surface . Misfolded proteins were retained within the ER lumen permanently or were transported to the cytosol in a retrograde manner and eventually degraded through proteasomes .…”

Section: Introductionmentioning

confidence: 99%

Asp68His mutation in the A1 domain of human factor V causes impaired secretion and ineffective translocation

et al. 2014

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Congenital factor V (FV) deficiency is a rare inherited disorder. We determined the mechanism of a missense mutation, Asp68His, in the A1 domain of the FV protein, is associated with severe FV deficiency. We characterized the mutant FV-Asp68His protein using in vitro expression studies by using specific secretion and degradation pathway inhibitors and analysed the intracellular translocation of the mutant protein by immunofluorescence staining. The Asp68His mutation caused very low levels of FV protein in the conditioned media, with normal specific FV activity. Similar mRNA degradation rates between FV-wild-type (wt) and FV-Asp68His mRNA showed that the Asp68His mutation does not affect FV expression at the transcriptional level. A specific secretion pathway inhibitor, brefeldin A, was used to demonstrate that the lower efficiency of transport to the outside of the cell for FV-Asp68His mutant protein compared with that of the FV-wt protein. Furthermore, we showed that the Asp68His mutation resulted in increased intracellular degradation through a MG132-mediated proteasomal degradation pathway. In the transfected cell lysates, FV-wt protein had multiple posttranslational modified forms, but the FV-Asp68His protein was not completely glycosylated. We further observed that the FV-Asp68His protein was retrieved in the endoplasmic reticulum only and did not undergo transport to the Golgi apparatus, leading to impaired secretion. These results strongly suggest that the Asp68His mutation may result in intracellular defective trafficking and enhanced degradation, and impaired secretion of FV protein.

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Saccharomyces cerevisiaeRot1 Is an Essential Molecular Chaperone in the Endoplasmic Reticulum

Takeuchi

Kohno

2008

MBoC

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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.

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Causal Links Between Protein Folding in the ER and Events Along the Secretory Pathway

Cited by 3 publications

References 25 publications

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Asp68His mutation in the A1 domain of human factor V causes impaired secretion and ineffective translocation

Saccharomyces cerevisiaeRot1 Is an Essential Molecular Chaperone in the Endoplasmic Reticulum

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