Kyohei Umebayashi

Intracellular Inclusions Containing Mutant α1-Antitrypsin Z Are Propagated in the Absence of Autophagic Activity

Mutant α1-antitrypsin Z (α1-ATZ) protein, which has a tendency to form aggregated polymers as it accumulates within the endoplasmic reticulum of the liver cells, is associated with the development of chronic liver injury and hepatocellular carcinoma in hereditary α1-antitrypsin (α1-AT) deficiency. Previous studies have suggested that efficient intracellular degradation of α1-ATZ is correlated with protection from liver disease in α1-AT deficiency and that the ubiquitin-proteasome system accounts for a major route, but not the sole route, of α1-ATZ disposal. Yet another intracellular degradation system, autophagy, has also been implicated in the pathophysiology of α1-AT deficiency. To provide genetic evidence for autophagy-mediated disposal of α1-ATZ, here we used cell lines deleted for the Atg5 gene that is necessary for initiation of autophagy. In the absence of autophagy, the degradation of α1-ATZ was retarded, and the characteristic cellular inclusions of α1-ATZ accumulated. In wild-type cells, colocalization of the autophagosomal membrane marker GFP-LC3 and α1-ATZ was observed, and this colocalization was enhanced when clearance of autophagosomes was prevented by inhibiting fusion between autophagosome and lysosome. By using a transgenic mouse with liver-specific inducible expression of α1-ATZ mated to the GFP-LC3 mouse, we also found that expression of α1-ATZ in the liver in vivo is sufficient to induce autophagy. These data provide definitive evidence that autophagy can participate in the quality control/degradative pathway for α1-ATZ and suggest that autophagic degradation plays a fundamental role in preventing toxic accumulation of α1-ATZ.

Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane

It was known that the uptake of tryptophan is reduced in the yeast erg6 mutant, which is defective in a late step of ergosterol biosynthesis. Here, we show that this is because the high affinity tryptophan permease Tat2p is not targeted to the plasma membrane. In wild-type cells, the plasma membrane localization of Tat2p is regulated by the external tryptophan concentration. Tat2p is transported from the Golgi apparatus to the vacuole at high tryptophan, and to the plasma membrane at low tryptophan. However, in the erg6 mutant, Tat2p is missorted to the vacuole at low tryptophan. The plasma membrane targeting of Tat2p is dependent on detergent-insoluble membrane domains, suggesting that sterol affects the sorting through the organization of lipid rafts. The erg6 mutation also caused missorting to the multivesicular body pathway in late endosomes. Thus, sterol composition is crucial for protein sorting late in the secretory pathway. Tat2p is subject to polyubiquitination, which acts as a vacuolar-targeting signal, and the inhibition of this process suppresses the Tat2p sorting defects of the erg6 mutant. The sorting mechanisms of Tat2p that depend on both sterol and ubiquitin will be discussed.

Ubc4/5 and c-Cbl continue to ubiquitinate EGF receptor after internalization to facilitate polyubiquitination and degradation

c-Cbl is the E3 ubiquitin ligase that ubiquitinates the epidermal growth factor (EGF) receptor (EGFR). On the basis of localization, knockdown, and in vitro activity analyses, we have identified the E2 ubiquitin-conjugating enzyme that cooperates with c-Cbl as Ubc4/5. Upon EGF stimulation, both Ubc4/5 and c-Cbl were relocated to the plasma membrane and then to Hrs-positive endosomes, strongly suggesting that EGFR continues to be ubiquitinated after internalization. Our time-course experiment showed that EGFR undergoes polyubiquitination, which seemed to be facilitated during the transport to Hrs-positive endosomes. Use of a conjugation-defective ubiquitin mutant suggested that receptor polyubiquitination is required for efficient interaction with Hrs and subsequent sorting to lysosomes. Abrupt inhibition of the EGFR kinase activity resulted in dissociation of c-Cbl from EGFR. Concomitantly, EGFR was rapidly deubiquitinated and its degradation was delayed. We propose that sustained tyrosine phosphorylation of EGFR facilitates its polyubiquitination in endosomes and counteracts rapid deubiquitination, thereby ensuring Hrs-dependent lysosomal sorting.

Roles of O-Mannosylation of Aberrant Proteins in Reduction of the Load for Endoplasmic Reticulum Chaperones in Yeast

The protein quality control system in the endoplasmic reticulum (ER) ensures that only properly folded proteins are deployed throughout the cells. When nonnative proteins accumulate in the ER, the unfolded protein response is triggered to limit further accumulation of nonnative proteins and the ER is cleared of accumulated nonnative proteins by the ER-associated degradation (ERAD). In the yeast ER, aberrant nonnative proteins are mainly directed for the ERAD, but a distinct fraction of them instead receive O-mannosylation. In order to test whether O-mannosylation might also be a mechanism to process aberrant proteins in the ER, here we analyzed the effect of O-mannosylation on two kinds of model aberrant proteins, a series of N-glycosylation site mutants of prepro-α-factor and a pro-region-deleted derivative of Rhizopus niveus aspartic proteinase-I (Δpro) both in vitro and in vivo. O-Mannosylation increases solubilities of the aberrant proteins and renders them less dependent on the ER chaperone, BiP, for being soluble. The release from ER chaperones allows the aberrant proteins to exit out of the ER for the normal secretory pathway transport. When the gene for Pmt2p, responsible for the O-mannosylation of these aberrant proteins, and that for the ERAD were simultaneously deleted, the cell exhibited enhanced unfolded protein response. O-Mannosylation may therefore function as a fail-safe mechanism for the ERAD by solubilizing the aberrant proteins that overflowed from the ERAD pathway and reducing the load for ER chaperones.

ACCUMULATION OF MISFOLDED PROTEIN AGGREGATES LEADS TO THE FORMATION OF RUSSELL BODY-LIKE DILATED ENDOPLASMIC RETICULUM IN YEAST

RNAP‐I, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its amino‐terminus. Our previous study indicated that the prosequence of RNAP‐I had important roles in its correct folding and secretion in yeast, and that a prosequence‐deleted derivative of RNAP‐I, Δpro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation of Δpro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that Δpro is recognized as an unfolded protein species in the ER. Our biochemical data demonstrated that Δpro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed that the Δpro aggregates were indeed visible as electron‐dense regions in the ER and nuclear envelope. Such ‘chaperone‐associated misfolded protein bodies’ were observed for the first time in yeast. Morphologies of the ER and nucleus were drastically altered by the accumulation of the Δpro aggregates. The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded Δpro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the Δpro aggregates are degraded.