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Functional redundancy of Rpn5 in budding yeast allows its participation and function in two distinct but structurally related PCI complexes: the proteasome lid and the CSN. As a lid subunit, Rpn5 stabilizes proteasome integrity; as a CSN subunit, Rpn5 is required for enzymatic hydrolysis of Rub1/Nedd8 from cullins., Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.

Protein aggregation negatively affects key enzymes of major metabolic pathways in mitochondria. The main mitochondrial chaperones Hsp70 and Hsp60 have a limited protective effect against aggregation. In contrast, the ATP-dependent matrix protease Pim1/LON significantly reduces aggregate levels by preventing the accumulation of damaged polypeptides., Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1-/- cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1-/- cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis. © 2011 Kolanczyk et al., published_or_final_version

This study reveals that components of the yeast ERAD-L pathway can discriminate between two subtly different forms of the same toxin substrate. Although precytosolic requirements are similar for both toxin structures, there is a divergence in fate on the cytosolic face of the ER membrane., We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTAΔ), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER–Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTAΔ, although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.

Molting of nematodes involves the synthesis and removal of a collagen-rich exoskeleton. We describe Caenorhabditis elegans MLT-10, which defines a large family of DUF644 and proline-rich repeat proteins. We show that MLT-10 is released from the epidermis during molting and that MLT-10 is involved in renewal of the exoskeleton and development of the epidermis., The molting cycle of nematodes involves the periodic synthesis and removal of a collagen-rich exoskeleton, but the underlying molecular mechanisms are not well understood. Here, we describe the mlt-10 gene of Caenorhabditis elegans, which emerged from a genetic screen for molting-defective mutants sensitized by low cholesterol. MLT-10 defines a large family of nematode-specific proteins comprised of DUF644 and tandem P-X2-L-(S/T)-P repeats. Conserved nuclear hormone receptors promote expression of the mlt-10 gene in the hypodermis whenever the exoskeleton is remade. Further, a MLT-10::mCherry fusion protein is released from the hypodermis to the surrounding matrices and fluids during molting. The fusion protein is also detected in strands near the surface of animals. Both loss-of-function and gain-of-function mutations of mlt-10 impede the removal of old cuticles. However, the substitution mutation mlt-10(mg364), which disrupts the proline-rich repeats, causes the most severe phenotype. Mutations of mlt-10 are also associated with abnormalities in the exoskeleton and improper development of the epidermis. Thus, mlt-10 encodes a secreted protein involved in three distinct but interconnected aspects of the molting cycle. We propose that the molting cycle of C. elegans involves the dynamic assembly and disassembly of MLT-10 and possibly the paralogs of MLT-10.

The yeast triacylglycerol (TAG) lipases Tgl3p and Tgl5p not only contain a TAG lipase, but also an acyltransferase motif. We show that purified Tgl3p and Tgl5p indeed exhibit lysophospholipid acyltransferase activity. Both Tgl3p and Tgl5p affect the level of glycerophospholipids, and Tgl3p is also important for efficient sporulation of yeast., In the yeast, mobilization of triacylglycerols (TAGs) is facilitated by the three TAG lipases Tgl3p, Tgl4p, and Tgl5p. Motif search analysis, however, indicated that Tgl3p and Tgl5p do not only contain the TAG lipase motif GXSXG but also an H-(X)4-D acyltransferase motif. Interestingly, lipid analysis revealed that deletion of TGL3 resulted in a decrease and overexpression of TGL3 in an increase of glycerophospholipids. Similar results were obtained with TGL5. Therefore, we tested purified Tgl3p and Tgl5p for acyltransferase activity. Indeed, both enzymes not only exhibited lipase activity but also catalyzed acylation of lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using variants of Tgl3p created by site-directed mutagenesis clearly demonstrated that the two enzymatic activities act independently of each other. We also showed that Tgl3p is important for efficient sporulation of yeast cells, but rather through its acyltransferase than lipase activity. In summary, our results demonstrate that yeast Tgl3p and Tgl5p play a dual role in lipid metabolism contributing to both anabolic and catabolic processes.

The mammalian ER contains 23 members of the PDI superfamily. Their substrate specificity is largely unknown. TMX1 shows a preference for membrane-bound, cysteine-containing polypeptides., The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. The lumen of the mammalian ER contains >20 members of the protein disulfide isomerase (PDI) superfamily, which ensure formation of the correct set of intramolecular and intermolecular disulfide bonds as crucial, rate-limiting reactions of the protein folding process. Components of the PDI superfamily may also facilitate dislocation of misfolded polypeptides across the ER membrane for ER-associated degradation (ERAD). The reasons for the high redundancy of PDI family members and the substrate features required for preferential engagement of one or the other are poorly understood. Here we show that TMX1, one of the few transmembrane members of the family, forms functional complexes with the ER lectin calnexin and preferentially intervenes during maturation of cysteine-containing, membrane-associated proteins while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane. As such, TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.

RIO proteins form a conserved family of atypical protein kinases. Humans possess three distinct RIO kinases—hRio1, hRio2, and hRio3, of which only hRio2 has been characterized with respect to its role in ribosomal biogenesis. Here we show that both hRio1 and hRio3, like hRio2, are associated with precursors of 40S ribosomal subunits in human cells. Furthermore, we demonstrate that depletion of hRio1 by RNA interference affects the last step of 18S rRNA maturation and causes defects in the recycling of several trans-acting factors (hEnp1, hRio2, hLtv1, hDim2/PNO1, and hNob1) from pre-40S subunits in the cytoplasm. Although the effects of hRio1 and hRio2 depletion are similar, we show that the two kinases are not fully interchangeable. Moreover, rescue experiments with a kinase-dead mutant of hRio1 revealed that the kinase activity of hRio1 is essential for the recycling of the endonuclease hNob1 and its binding partner hDim2 from cytoplasmic pre-40S. Kinase-dead hRio1 is trapped on pre-40S particles containing hDim2 and hNob1 but devoid of hEnp1, hLtv1, and hRio2. These data reveal a role of hRio1 in the final stages of cytoplasmic pre-40S maturation., Molecular Biology of the Cell, 23 (1), ISSN:1939-4586, ISSN:1059-1524

The yeast cytochrome oxidase Cox3p assembly module is shown to consist of Cox3p, Cox4p, Cox7p, Cox13p, and accessory factor Rcf1p. The results support an assembly model in which three modules, each containing one of the three core subunits and a unique subset of nuclear-derived subunits, interact to form the holoenzyme., Yeast cytochrome oxidase (COX) was previously inferred to assemble from three modules, each containing one of the three mitochondrially encoded subunits and a different subset of the eight nuclear gene products that make up this respiratory complex. Pull-down assays of pulse-labeled mitochondria enabled us to characterize Cox3p subassemblies that behave as COX precursors and contain Cox4p, Cox7p, and Cox13p. Surprisingly, Cox4p is a constituent of two other complexes, one of which was previously proposed to be an intermediate of Cox1p biogenesis. This suggests that Cox4p, which contacts Cox1p and Cox3p in the holoenzyme, can be incorporated into COX by two alternative pathways. In addition to subunits of COX, some Cox3p intermediates contain Rcf1p, a protein associated with the supercomplex that stabilizes the interaction of COX with the bc1 (ubiquinol-cytochrome c reductase) complex. Finally, our results indicate that although assembly of the Cox1p module is not contingent on the presence of Cox3p, the converse is not true, as none of the Cox3p subassemblies were detected in a mutant blocked in translation of Cox1p. These studies support our proposal that Cox3p and Cox1p are separate assembly modules with unique compositions of ancillary factors and subunits derived from the nuclear genome.