Characterization of Saccharomyces cerevisiae acyl-protein thioesterase 1, the enzyme responsible for G protein alpha subunit deacylation in vivo

Thioacylation is a reversible lipid modification of proteins that plays a role in the regulation of signal transduction. Acyl-protein thioesterase 1 (APT1) was identified as an enzyme capable of deacylating some thioacylated proteins in vitro. Saccharomyces cerevisiae open reading frame YLR118c encodes an enzyme homologous to Rattus norvegicus APT1. We demonstrate that the catalytic activity of the protein encoded by the yeast open reading frame is similar to that of rat APT1, and we designate the protein S. cerevisiae Apt1p. Yeasts bearing a disruption of the APT1 gene lack significant biochemically detectable acyl-protein thioesterase activity. They also fail to deacylate Gpa1p, the yeast G alpha subunit, in metabolic radiolabeling studies. We conclude that native APT1 is the enzyme responsible for G alpha subunit deacylation in S. cerevisiae and presumably other eukaryotes as well.     

Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes

We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.     

Rad54, the motor of homologous recombination

Homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. The invasion produces a cross-stranded structure, known as the Holliday junction. Here, we describe the properties of Rad54, an important and versatile HR protein that is evolutionarily conserved in eukaryotes. Rad54 is a motor protein that translocates along dsDNA and performs several important functions in HR. The current review focuses on the recently identified Rad54 activities which contribute to the late phase of HR, especially the branch migration of Holliday junctions.     

Differences in the DNA replication of unicellular eukaryotes and metazoans: known unknowns

Although the basic mechanisms of DNA synthesis are conserved across species, there are differences between simple and complex organisms. In contrast to lower eukaryotes, replication origins in complex eukaryotes lack DNA sequence specificity, can be activated in response to stressful conditions and require poorly conserved factors for replication firing. The response to replication fork damage is monitored by conserved proteins, such as the TIPIN–TIM–CLASPIN complex. The absence of this complex induces severe effects on yeast replication, whereas in higher eukaryotes it is only crucial when the availability of replication origins is limiting. Finally, the dependence of DNA replication on homologous recombination proteins such as RAD51 and the MRE11–RAD50–NBS1 complex is also different; they are dispensable for yeast S‐phase but essential for accurate DNA replication in metazoans under unchallenged conditions. The reasons for these differences are not yet understood. Here, we focus on some of these known unknowns of DNA replication.     

Structure and expression of the Zea mays mutS-homologs Mus1 and Mus2

DNA mismatch repair proteins play an important role in maintaining the integrity of the genetic information during replication and homologous recombination. The MutS-homologous (MSH) and MutL-homologous (MLH) proteins are highly conserved among all prokaryotes and eukaryotes. We have isolated two mutS homologous genes from Zea mays, named Mus1 and Mus2. Phylogenetic analysis identifies Mus1 as a member of the MSH2 protein family. Mus2 is an ortholog of the Arabidopsis thaliana MSH7 protein and belongs to a subgroup of MSH proteins that is possibly plant-specific. Mus1 and Mus2 are expressed at very low levels. Mus1 is located on chromosome 7L near locus b32B, and mus2 maps on chromosome 3S.     

Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins

Mitochondria are indispensable for cell viability; however, major mitochondrial functions including citric acid cycle and oxidative phosphorylation are dispensable. Most known essential mitochondrial proteins are involved in preprotein import and assembly, while the only known essential biosynthetic process performed by mitochondria is the biogenesis of iron-sulfur clusters (ISC). The components of the mitochondrial ISC-assembly machinery are derived from the prokaryotic ISC-assembly machinery. We have identified an essential mitochondrial matrix protein, Isd11 (YER048w-a), that is found in eukaryotes only. Isd11 is required for biogenesis of cellular Fe/S proteins and thus is a novel subunit of the mitochondrial ISC-assembly machinery. It forms a complex with the cysteine desulfurase Nfs1 and is required for formation of an Fe/S cluster on the Isu scaffold proteins. We conclude that Isd11 is an indispensable eukaryotic component of the mitochondrial machinery for biogenesis of Fe/S proteins.     

The GreenCut2 resource, a phylogenomically derived inventory of proteins specific to the plant lineage

The plastid is a defining structure of photosynthetic eukaryotes and houses many plant-specific processes, including the light reactions, carbon fixation, pigment synthesis, and other primary metabolic processes. Identifying proteins associated with catalytic, structural, and regulatory functions that are unique to plastid-containing organisms is necessary to fully define the scope of plant biochemistry. Here, we performed phylogenomics on 20 genomes to compile a new inventory of 597 nucleus-encoded proteins conserved in plants and green algae but not in non-photosynthetic organisms. 286 of these proteins are of known function, whereas 311 are not characterized. This inventory was validated as applicable and relevant to diverse photosynthetic eukaryotes using an additional eight genomes from distantly related plants (including Micromonas, Selaginella, and soybean). Manual curation of the known proteins in the inventory established its importance to plastid biochemistry. To predict functions for the 52% of proteins of unknown function, we used sequence motifs, subcellular localization, co-expression analysis, and RNA abundance data. We demonstrate that 18% of the proteins in the inventory have functions outside the plastid and/or beyond green tissues. Although 32% of proteins in the inventory have homologs in all cyanobacteria, unexpectedly, 30% are eukaryote-specific. Finally, 8% of the proteins of unknown function share no similarity to any characterized protein and are plant lineage-specific. We present this annotated inventory of 597 proteins as a resource for functional analyses of plant-specific biochemistry.     

Unique invasions and resolutions: DNA repair proteins in meiotic recombination in Drosophila melanogaster

To ensure the accurate disjunction of homologous chromosomes during meiosis, most eukaryotes rely on physical connections called chiasmata, which form at sites of crossing over. In the absence of crossing over, homologs may segregate randomly, resulting in high frequencies of aneuploid gametes. The process of meiotic recombination poses unique problems for the cell that must be overcome to ensure normal disjunction of homologous chromosomes. How is it ensured that crossovers occur between homologous chromosomes, rather than between sister chromatids? What determines the number and location of crossovers? The functions of DNA repair proteins hold some of the answers to these questions. In this review, we discuss DNA repair proteins that function in meiotic recombination in Drosophila melanogaster. We emphasize the processes of strand invasion and Holliday junction resolution in order to shed light on the questions raised above. Also, we compare the variety of ways several eukaryotes perform these processes and the different proteins they require.     

S1 and S2, the linear mitochondrial DNAs present in a male sterile line of maize, possess terminally attached proteins.

S1 and S2 are short linear mitochondrial DNA molecules found in a particular male sterile cytoplasm of maize. We show here that these DNA molecules and two other related linear DNA species found in maize mitochondria, have proteins attached, probably covalently, to their 5' ends. This is the first demonstration of such a linear DNA-terminal protein association in higher eukaryotes. Such proteins may be involved in priming replication of these DNAs.     

Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins

The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1 , a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA . The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum cofactor locus chl6 . As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E. coli Moco protein MoeA, the C-terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli , is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.     

Interchanging Functionality Among Homologous Elongation Factors Using Signatures of Heterotachy

Numerous models of molecular evolution have been formulated to describe the forces that shape sequence divergence among homologous proteins. These models have greatly enhanced our understanding of evolutionary processes. Rarely are such models empirically tested in the laboratory, and even more rare, are such models exploited to generate novel molecules useful for synthetic biology. Here, we experimentally demonstrate that the heterotachy model of evolution captures signatures of functional divergence among homologous elongation factors (EFs) between bacterial EF-Tu and eukaryotic eEF1A. These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome. Upon release from the ribosome, the EFs are recharged by nucleotide exchange factors EF-Ts in bacteria or eEF1B in eukaryotes. The two nucleotide exchange factors perform analogous functions despite not being homologous proteins. The heterotachy model was used to identify a set of sites in eEF1A/EF-Tu associated with eEF1B binding in eukaryotes and another reciprocal set associated with EF-Ts binding in bacteria. Introduction of bacterial EF-Tu residues at these sites into eEF1A protein efficiently disrupted binding of cognate eEF1B as well as endowed eEF1A with the novel ability to bind bacterial EF-Ts. We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system.     

Smc5/6 is required for repair at collapsed replication forks

In eukaryotes, three pairs of structural-maintenance-of-chromosome (SMC) proteins are found in conserved multisubunit protein complexes required for chromosomal organization. Cohesin, the Smc1/3 complex, mediates sister chromatid cohesion while two condensin complexes containing Smc2/4 facilitate chromosome condensation. Smc5/6 scaffolds an essential complex required for homologous recombination repair. We have examined the response of smc6 mutants to the inhibition of DNA replication. We define homologous recombination-dependent and -independent functions for Smc6 during replication inhibition and provide evidence for a Rad60-independent function within S phase, in addition to a Rad60-dependent function following S phase. Both genetic and physical data show that when forks collapse (i.e., are not stabilized by the Cds1Chk2 checkpoint), Smc6 is required for the effective repair of resulting lesions but not for the recruitment of recombination proteins. We further demonstrate that when the Rad60-dependent, post-S-phase Smc6 function is compromised, the resulting recombination-dependent DNA intermediates that accumulate following release from replication arrest are not recognized by the G2/M checkpoint.