We are studying endoplasmic reticulum-associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI 332 , containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI 332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ϳ45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI 332 in which the single used N-glycosylation consensus site had been removed (RI 332 -Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI 332 degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI 332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI 332 . After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI 332 and RI 332 -Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI 332 and RI 332 -Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI 332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives. INTRODUCTIONNewly synthesized proteins of the endomembrane system and most secreted proteins of eukaryotic cells enter the secretory pathway through the translocation channel at the membrane of the endoplasmic reticulum (ER). The lumen of the ER is the site where translocated proteins assume their secondary structure and where assembly of oligomeric complexes occurs. Additionally, newly synthesized proteins undergo cotranslational and posttranslational modifications in the lumen of the ER, some of which allow transient interactions of the folding polypeptide chains with a set of ER-resident proteins (Hammond and Helenius, 1995;Leitzgen and Haas, 1998). Only after acquiring a fully folded, native conformation can proteins complete their journey through the secretory pathPresent addresses:† Dipartimento Medicina Sperimentale e Diagnostica, Sezione di Patologia Generale, Universitá di Ferrara, Ferrara, Italy; ‡ Spiegelmayr K...

BackgroundThe pivotal role of mitochondria in energy production and free radical generation suggests that the mitochondrial genome could have an important influence on the expression of multifactorial age related diseases. Substitution of T to C at nucleotide position 16189 in the hypervariable D-loop of the control region (CR) of mitochondrial DNA (mtDNA) has attracted research interest because of its suspected association with various multifactorial diseases. The aim of the present study was to compare the frequency of this polymorphism in the CR of mtDNA in patients with coronary artery disease (CAD, n = 482) and type 2 diabetes mellitus (T2DM, n = 505) from two study centers, with healthy individuals (n = 1481) of Middle European descent in Austria.Methodology and Principal FindingsCR polymorphisms and the nine major European haplogroups were identified by DNA sequencing and primer extension analysis, respectively. Frequencies and Odds Ratios for the association between cases and controls were calculated. Compared to healthy controls, the prevalence of T16189C was significantly higher in patients with CAD (11.8% vs 21.6%), as well as in patients with T2DM (11.8% vs 19.4%). The association of CAD, but not the one of T2DM, with T16189C remained highly significant after correction for age, sex and body mass index (BMI) and was independent of the two study centers.Conclusions and SignificanceOur results show for the first time a significant association of T16189C with CAD in a Middle European population. As reported in other studies, in patients with T2DM an association with T16189C in individuals of European decent remains questionable.

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.