A yeast mitochondrial membrane methyltransferase-like protein can compensate for oxa1 mutations

Members of the Oxa1p/Alb3/YidC family mediate the insertion of various organelle or bacterial hydrophobic proteins into membranes. They present at least five transmembrane segments (TM) linked by hydrophilic domains located on both sides of the membrane. To examine how Oxa1p structure relates to its function, we have introduced point mutations and large deletions into various domains of the yeast mitochondrial protein. These mutants allowed us to show the importance of the first TM domain as well as a synergistic interaction between the first loop and the C-terminal tail, which both protrude into the matrix. These mutants also led to the isolation of a high copy suppressor, OMS1, which encodes a member of the methyltransferase family. Overexpression of OMS1 seems to increase the steady-state level of both the mutant and wild-type Oxa1p. We show that Oms1p is a mitochondrial inner membrane protein inserted independently of Oxa1p. Oms1p presents one TM and a N-in C-out topology with the C-terminal domain carrying the methyltransferase-like domain. A conserved motif within this domain is essential for the suppression of oxa1 mutations. We discuss the possible role of Oms1p on Oxa1p intermembrane space domain.     

Virtual identification of essential proteins within the protein interaction network of yeast

Topological analysis of large scale protein-protein interaction networks (PINs) is important for understanding the organizational and functional principles of individual proteins. The number of interactions that a protein has in a PIN has been observed to be correlated with its indispensability. Essential proteins generally have more interactions than the nonessential ones. We show here that the lethality associated with removal of a protein from the yeast proteome correlates with different centrality measures of the nodes in the PIN, such as the closeness of a protein to many other proteins, or the number of pairs of proteins which need a specific protein as an intermediary in their communications, or the participation of a protein in different protein clusters in the PIN. These measures are significantly better than random selection in identifying essential proteins in a PIN. Centrality measures based on graph spectral properties of the network, in particular the subgraph centrality, show the best performance in identifying essential proteins in the yeast PIN. Subgraph centrality gives important structural information about the role of individual proteins, and permits the selection of possible targets for rational drug discovery through the identification of essential proteins in the PIN.     

The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex

In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)-binding protein I (Pab1p). We report the characterization of Pfs2p, a WD-repeat protein previously identified in a multiprotein complex carrying PF I-Pap1p activity. The 3'-end-processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation. With a one-step affinity purification method that exploits protein A-tagged Pfs2p, we showed that this protein is part of a CF II-PF I complex. Pull-down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II-PF I and CF IA. These results show that Pfs2p plays an essential role in 3'-end formation by bridging different processing factors and thereby promoting the assembly of the processing complex.     

Fta2, an essential fission yeast kinetochore component, interacts closely with the conserved Mal2 protein

The fission yeast multiprotein-component Sim4 complex plays a fundamental role in the assembly of a functional kinetochore. It affects centromere association of the histone H3 variant CENP-A as well as kinetochore association of the DASH complex. Here, multicopy suppressor analysis of a mutant version of the Sim4 complex component Mal2 identified the essential Fta2 kinetochore protein, which is required for bipolar chromosome attachment. Kinetochore localization of Mal2 and Fta2 depends on each other, and overexpression of one protein can rescue the phenotype of the mutant version of the other protein. fta2 mal2 double mutants were inviable, implying that the two proteins have an overlapping function. This close interaction with Fta2 is not shared by other Sim4 complex components, indicating the existence of functional subgroups within this complex. The Sim4 complex seems to be assembled in a hierarchical way, because Fta2 is localized correctly in a sim4 mutant. However, Fta2 kinetochore localization is reduced in a spc7 mutant. Spc7, a suppressor of the EB1 family member Mal3, is part of the conserved Ndc80-MIND-Spc7 kinetochore complex.     

A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole

The VPS15 gene encodes a novel protein kinase homolog that is essential for the efficient delivery of soluble hydrolases to the yeast vacuole. Point mutations altering highly conserved residues within the Vps15p kinase domain result in the secretion of multiple vacuolar proteases. In addition, the in vivo phosphorylation of Vps15p is defective in these kinase domain mutants, suggesting that Vps15p may regulate specific protein phosphorylation reactions required for protein sorting to the yeast vacuole. Subcellular fractionation studies further demonstrate that the 1455 amino acid Vps15p is peripherally associated with the cytoplasmic face of a late Golgi or vesicle compartment. This association may be mediated by myristate as Vps15p contains a consensus signal for N-terminal myristoylation. We propose that protein phosphorylation may act as a molecular "switch" within intracellular protein sorting pathways by actively diverting proteins from a default transit pathway (e.g., secretion) to an alternative pathway (e.g., to the vacuole).     

Role of the alfalfa mosaic virus methyltransferase-like domain in negative-strand RNA synthesis

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.     

Ege A,a novel C2 domain containing protein,is essential for GPCR-mediated gene expression in dictyostelium

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.     

The PH domain of the yeast GEF Rom2p serves an essential function in vivo

In a screen designed to identify new upstream components of the Pkc1p-MAP kinase signal transduction pathway that responds to cell wall damage in yeast, we identified a new mutant allele of the ROM2 gene, which encodes a GDP/GTP exchange factor that acts on Rho1p. This allele, originally termed ubk1 (upstream of Bck1p) encodes a truncated protein that lacks the putative PH domain. Complementation experiments showed that genes coding for several known components of the pathway are able to suppress the ubk1 mutation to various degrees when introduced on low- or high-copy-number vectors. Analysis of several rom2 mutants showed that mutants in which the PH domain is deleted result in a phenotype indistinguishable from that of a strain deleted for the entire gene, indicating that this domain fulfills an essential function in vivo. Furthermore, we found that the growth phenotype of rom2 mutants is highly dependent on the strain background. Surprisingly, analysis of the phosphorylation status of Mpk1p in these mutants showed an elevated level of doubly phosphorylated Mpk1 protein, indicating that the growth defect of rom2 mutants is not due to an inability to activate the MAP kinase module, but rather to lack of a function of the Rom2 protein that has yet to be identified precisely.     

A genetic method to enhance the accumulation of S-adenosylmethionine in yeast

S-Adenosylmethionine (SAM) is a key component of sulphur amino acid metabolism in living organisms and is synthesised from methionine and adenosine triphosphate by methionine adenosyltransferase. This molecule serves as the main biological methyl donor due to its active methylthio ether group. Notably, SAM has shown beneficial effects in clinical trials for the treatment of alcoholic liver disease, depression and joint pain. Due to the high potential value of SAM, current research efforts are attempting to develop a more rapid, cost-effective and higher yielding SAM production method than the conventional production system. In this mini-review, we describe the previously reported yeast gene that contributes to SAM accumulation by overexpression, mutation or deletion and summarise the genetic approach for the production of SAM in large industrial quantities.

     

Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis

In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Δmrs3 and Δmrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX₂CXC and twin CX₂C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Δdre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.     

Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle

The activities of type 1 protein phosphatase (PP1) and 2A (PP2A) have distinct, essential roles in cell cycle control. Two previously identified PP1 genes (dis2+ and sds21+) and two PP2A genes (ppa1+ and ppa2+), highly homologous to mammalian PP2A, have been isolated from fission yeast. Only double gene disruption of both PP2A genes results in lethality, as is the case for PP1 genes. By fractionating and assaying PPases in wild-type, various deletion, and point mutant strains, the decrease of PP1 or PP2A activity is shown to cause mitotic defects, exhibiting strikingly different cell cycle phenotypes: cold-sensitive mutations in the same amino acid lesion of PP1 and PP2A produce chromosome nondisjunction and premature mitosis, respectively. Consistently, PP1 and PP2A genes cannot be functionally substituted. Although the overall levels of PP1 and PP2A activities do not fluctuate during the cell cycle, subpopulations might be regulated.     

The cyclin-dependent kinase inhibitory domain of the yeast Sic1 protein is contained within the C-terminal 70 amino acids

By inhibiting the activity of Cdc28/Clb cyclin-dependent protein kinase (CDK) complexes, Sic1 prevents the premature initiation of S phase in the yeast Saccharomyces cerevisiae. By testing a series of Sic1 truncation mutants, we have mapped the minimal domain necessary for Cdc28/Clb inhibition in vivo to the C-terminal 70 amino acids of Sic1. Site-directed mutagenesis was used to show that a sequence that matches the zRxL motif found in mammalian CDK inhibitors is essential for Sic1 function. This motif is not found in the Schizosaccharomyces CDK inhibitor p25^rum1, which appears to be a structural and functional homolog of Sic1. Based on the mutational data and sequence comparisons, we argue that Sic1 and p25^rum1 are structurally distinct from the known mammalian CDK inhibitors, but may bind CDK complexes in a manner more closely resembling CDK substrates like the retinoblastoma and E2F proteins. Received: 3 February 1999 / Accepted: 23 April 1999     

Self-association of the amino-terminal domain of the yeast TATA-binding protein

The amino-terminal domain of yeast TATA-binding protein has been proposed to play a crucial role in the self-association mechanism(s) of the full-length protein. Here we tested the ability of this domain to self-associate under a variety of solution conditions. Escherichia coli two-hybrid assays, in vitro pull-down assays, and in vitro cross-linking provided qualitative evidence for a limited and specific self-association. Sedimentation equilibrium analysis using purified protein was consistent with a monomer-dimer equilibrium with an apparent dissociation constant of approximately 8.4 microM. Higher stoichiometry associations remain possible but could not be detected by any of these methods. These results demonstrate that the minimal structure necessary for amino-terminal domain self-association must be present even in the absence of carboxyl-terminal domain structures. On the basis of these results we propose that amino-terminal domain structures contribute to the oligomerization interface of the full-length yeast TATA-binding protein.