Activation of the Ras-cAMP signal transduction pathway inhibits the proteasome-independent degradation of misfolded protein aggregates in the endoplasmic reticulum lumen.

Kyohei Umebayashi,Hiroyuki Horiuchi,Masamichi Takagi,Aiko Hirata,Akinori Ohta,Ryouichi Fukuda,Akihiko Nakano

doi:10.1074/jbc.m105829200

Kyohei Umebayashi, Hiroyuki Horiuchi + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m105829200

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Abstract

Many kinds of misfolded secretory proteins are known to be degraded in the endoplasmic reticulum (ER). Dislocation of misfolded proteins from the ER to the cytosol and subsequent degradation by the proteasome have been demonstrated. Using the yeast Saccharomyces cerevisiae, we have been studying the secretion of a heterologous protein, Rhizopus niveus aspartic proteinase-I (RNAP-I). Previously, we found that the pro sequence of RNAP-I is important for the folding and secretion, and that Deltapro, a mutated derivative of RNAP-I in which the entire region of the pro sequence is deleted, forms gross aggregates in the yeast ER. In this study, we show that the degradation of Deltapro occurs independently of the proteasome. Its degradation was not inhibited either by a potent proteasome inhibitor or in a proteasome mutant. We also show that neither the export from the ER nor the vacuolar proteinase is required for the degradation of Deltapro. These results raise the possibility that the Deltapro aggregates are degraded in the ER lumen. We have isolated a yeast mutant in which the degradation of Deltapro is delayed. We show that the mutated gene is IRA2, which encodes a GTPase-activating protein for Ras. Because Ira2 protein is a negative regulator of the Ras-cAMP pathway, this result suggests that hyperactivation of the Ras-cAMP pathway inhibits the degradation of Deltapro. Consistently, down-regulation of the Ras-cAMP pathway in the ira2 mutant suppressed the defect of the degradation of Deltapro. Thus, the Ras-cAMP signal transduction pathway seems to control the proteasome-independent degradation of the ER misfolded protein aggregates.

Highlights

Many kinds of misfolded secretory proteins are known to be degraded in the endoplasmic reticulum (ER)
In that study, ⌬pro was produced by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter and the dilation of the ER accompanying the accumulation of the ⌬pro aggregates was not remarkable, as judged from indirect immunofluorescence [34]
In ⌬pep4 prb1 cells, like wild-type cells, the accumulated ⌬pro was degraded after its production was shut off, and little was remaining after 8 h (Fig. 1A), indicating that vacuolar proteinases do not participate in the degradation of ⌬pro

Summary

EXPERIMENTAL PROCEDURES

Strains and Media—Escherichia coli strains XL1-Blue (recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac [FЈ proAB lacIqZ⌬M15 Tn10 (tetr)]) and HB101 (supE44 hsdS20 (rBϪ mBϪ) recA13 ara proA2 lacY1 galK2 rpsL20 xyl mti leuB6 thi1) were used as hosts for plasmid construction and propagation. Plasmids pYPR3841 and pYPR3841U, with which ⌬pro is expressed under the control of the GAL1 promoter and the GAPDH terminator, were described before [37, 38]. The 1.8-kb BamHI-HindIII fragment, which contained the GAL1 promoter, the ⌬pro coding region, and the GAPDH terminator, was prepared from pYPR3841. This fragment was inserted between the BamHI and HindIII sites of pBluescript II KSϩ, and re-isolated by digestion with BamHI and XhoI. Cells were collected and washed once, each by centrifugation for 1 min, and resuspended in 0.6 ml of the chase medium (YNBGal medium supplemented with 2% of casamino acids, 75 mM methionine, and 75 mM cysteine). Thin sections were incubated with the anti-RNAP-I antibody and subsequently with 15-nm gold particle-conjugated secondary antibody as described previously [37]

RESULTS

Having established that the genetic locus involved in the

DISCUSSION