Mdp1, a Saccharomyces Cerevisiae Gene Involved in Mitochondrial/Cytoplasmic Protein Distribution, Is Identical to the Ubiquitin-Protein Ligase Gene Rsp5

Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression. Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton. MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization. We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases. In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins.     

The growth of mdp1/rsp5 mutants of Saccharomyces cerevisiae is affected by mutations in the ATP-binding domain of the plasma membrane H+ -ATPase

Mutations in the PMA1 gene, encoding plasma membrane H+ -ATPase, were isolated that are able to suppress the temperature sensitivity (ts) phenotype of mdp1 mutations located in RSP5, the ubiquitin-protein ligase gene. The mdp1 mutants were previously found to change the mitochondrial/cytosolic distribution of Mod5p-I, the tRNA modifying enzyme, and to affect fluid phase endocytosis. The data presented reveal that mdp1 mutants are also pH sensitive, and hypersensitive to hygromycin B and paromomycin. The ts phenotype, hygromycin B and paromomycin sensitivity are suppressed by pmal-t, but the pH sensitivity, the effect of mdp1 on Mod5p-I cytoplasmic/mitochondrial localization and endocytosis are not. Characterization of pmal-t revealed the substitution of amino acid G(653)V in the ATP-binding domain of the H+ -ATPase. Our results indicate that Rsp5 ubiquitin-protein ligase may also influence, in addition to protein distribution, the functioning of plasma membrane H+ -ATPase and the response of cells to stress.     

Chaperone receptors: guiding proteins to intracellular compartments

Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519–530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529–535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763–2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41–50, 2003; Qbadou et al., EMBO J 25:1836–1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763–2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.     

Catch me if you can! Oxidative protein trapping in the intermembrane space of mitochondria

The intermembrane space (IMS) of mitochondria, the compartment that phylogenetically originated from the periplasm of bacteria, contains machinery to catalyze the oxidative folding of proteins (Mesecke, N., N. Terziyska, C. Kozany, F. Baumann, W. Neupert, K. Hell, and J.M. Herrmann. 2005. Cell. 121:1059-1069; Rissler, M., N. Wiedemann, S. Pfannschmidt, K. Gabriel, B. Guiard, N. Pfanner, and A. Chacinska. 2005. J. Mol. Biol. 353: 485-492; Tokatlidis, K. 2005. Cell. 121:965-96). This machinery introduces disulfide bonds into newly imported precursor proteins, thereby locking them in a folded conformation. Because folded proteins cannot traverse the translocase of the outer membrane, this stably traps the proteins in the mitochondria. The principle of protein oxidation in the IMS presumably has been conserved from the bacterial periplasm and has been adapted during evolution to drive the vectorial translocation of proteins from the cytosol into the mitochondria.     

Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase.

Selective degradation by proteasomes of ornithine decarboxylase, the initial enzyme in polyamine biosynthesis, is mediated by the polyamine-inducible protein antizyme. Antizyme binds to a region near the N terminus of ornithine decarboxylase (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-3562, 1992). This interaction induces a conformational change in ornithine decarboxylase that exposes its C terminus and inactivates the enzyme (X. Li and P. Coffino, Mol. Cell. Biol. 13:1487-1492, 1993). Here we show that the C-terminal half of antizyme alone can inactivate ornithine decarboxylase and alter its conformation, but it cannot direct degradation of the enzyme, either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote degradation.     

Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase

To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.     

Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae.

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which are encoded by the CKA1 and CKA2 genes, respectively. Null mutations in the CKA1 gene do not confer a detectable phenotype (J. L.-P. Chen-Wu, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 8:4981-4990, 1988), presumably because of the presence of the CKA2 gene. We report here the cloning, sequencing, and disruption of the CKA2 gene. The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A. Saxena, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 7:3409-3417, 1987). Deletions of the CKA2 gene were constructed by gene replacement techniques. Haploid cells in which the CKA2 gene alone is disrupted show no detectable phenotype, but haploid cells carrying disruptions in both the CKA1 and CKA2 genes are inviable. Cells in which casein kinase II activity is depleted increase substantially in size prior to growth arrest, and a significant fraction of the arrested cells exhibit a pseudomycelial morphology. Disruption of the activity also results in flocculation. Yeast strains lacking both endogenous catalytic subunit genes can be rescued by expression of the alpha and beta subunits of Drosophila casein kinase II or by expression of the Drosophila alpha subunit alone, suggesting that casein kinase II function has been conserved through evolution.     

Human U1 small nuclear RNA pseudogenes do not map to the site of the U1 genes in 1p36 but are clustered in 1q12-q22.

Human U1 small nuclear RNA is encoded by approximately 30 gene copies. All of the U1 genes share several kilobases of essentially perfect flanking homology both upstream and downstream from the U1 coding region, but remarkably, for many U1 genes excellent flanking homology extends at least 24 kilobases upstream and 20 kilobases downstream. Class I U1 RNA pseudogenes are abundant in the human genome. These pseudogenes contain a complete but imperfect U1 coding region and possess extensive flanking homology to the true U1 genes. We mapped four class I pseudogenes by in situ hybridization to the long arm of chromosome 1, bands q12-q22, a region distinct from the site on the distal short arm of chromosome 1 to which the U1 genes have been previously mapped (Lund et al., Mol. Cell. Biol. 3:2211-2220, 1983; Naylor et al., Somat. Cell Mol. Genet. 10:307-313, 1984). We confirmed our in situ hybridization results by genomic blotting experiments with somatic cell hybrid lines with translocation products of human chromosome 1. These experiments provide further evidence that class I U1 pseudogenes and the true U1 genes are not interspersed. The results, along with those published elsewhere (Bernstein et al., Mol. Cell. Biol. 5:2159-2171, 1985), suggest that gene amplification may be responsible for the sequence homogeneity of the human U1 gene family.     

A codon change in beta-tubulin which drastically affects microtubule structure in Drosophila melanogaster fails to produce a significant phenotype in Saccharomyces cerevisiae.

The relative uniformity of microtubule ultrastructure in almost all eukaryotic cells is thought to be a consequence of the conserved elements of tubulin sequence. In support of this idea, a mutation in a beta-tubulin gene of Drosophila melanogaster, occurring at a highly conserved position, produces U-shaped microtubules, suggesting a defect in either nucleation or packing during assembly (M. T. Fuller, J. H. Caulton, J. A. Hutchens, T. C. Kaufman, and E. C. Raff, J. Cell Biol. 104:385-394, 1987, and J. E. Rudolph, M. Kimble, H. D. Hoyle, M. A. Subler, and E. C. Raff, Mol. Cell. Biol. 7:2231-2242, 1987). Surprisingly, we find that introducing the same mutation into the sole beta-tubulin gene of Saccharomyces cerevisiae has virtually no consequences for microtubule structure or function in that organism.