A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.     

Purification,characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae

Background

In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS).

Methods

The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant.

Results

The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14 kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30 °C and 7.0 respectively. The K_m V_max and K_cat against S-adenosyl-l-methionine (AdoMet) were 4.95 μM, 3.2 U/mg and 6.4 s^− 1 respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a K_i as 10.9 μM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kan^r exhibited almost 50% reduction in intracellular trehalose concentration.

Conclusion

A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae.

General significance

This is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.     

PHYSICO: An UNIX based Standalone Procedure for Computation of Individual and Group Properties of Protein Sequences

In the genomic and proteomic era, efficient and automated analyses of sequence properties of protein have become an important task in bioinformatics. There are general public licensed (GPL) software tools to perform a part of the job. However, computations of mean properties of large number of orthologous sequences are not possible from the above mentioned GPL sets. Further, there is no GPL software or server which can calculate window dependent sequence properties for a large number of sequences in a single run. With a view to overcome above limitations, we have developed a standalone procedure i.e. PHYSICO, which performs various stages of computation in a single run based on the type of input provided either in RAW-FASTA or BLOCK-FASTA format and makes excel output for: a) Composition, Class composition, Mean molecular weight, Isoelectic point, Aliphatic index and GRAVY, b) column based compositions, variability and difference matrix, c) 25 kinds of window dependent sequence properties. The program is fast, efficient, error free and user friendly. Calculation of mean and standard deviation of homologous sequences sets, for comparison purpose when relevant, is another attribute of the program; a property seldom seen in existing GPL softwares.

Availability

PHYSICO is freely available for non-commercial/academic user in formal request to the corresponding author ni.ca.vinurub.hcetoib@eejrenabka     

Three Residues of the Interdomain Linker Determine the Conformation and Single-base Deletion Fidelity of Y-family Translesion Polymerases

Dpo4 and Dbh are from two closely related Sulfolobus species and are well studied archaeal homologues of pol IV, an error prone Y-family polymerase from Escherichia coli. Despite sharing 54% amino acid identity, these polymerases display distinct mutagenic and translesion specificities. Structurally, Dpo4 and Dbh adopt different conformations because of the difference in relative orientation of their N-terminal catalytic and C-terminal DNA binding domains. Using chimeric constructs of these two polymerases, we have previously demonstrated that the interdomain linker is a major determinant of polymerase conformation, base-substitution fidelity, and abasic-site translesion synthesis. Here we find that the interdomain linker also affects the single-base deletion frequency and the mispair extension efficiency of these polymerases. Exchanging just three amino acids in the linkers of Dbh and Dpo4 is sufficient to change the fidelity by up to 30-fold, predominantly by altering the rate of correct (but not incorrect) nucleotide incorporation. Additionally, from a 2.4 Å resolution crystal structure, we have found that the three linker amino acids from Dpo4 are sufficient to allow Dbh to adopt the standard conformation of Dpo4. Thus, a small region of the interdomain linker, located more than 11 Å away from the catalytic residues, determines the fidelity of these Y-family polymerases, by controlling the alignment of substrates at the active site.     

Biomethanation of a cellulose-based substrate in the presence and absence of a cellulolytic bacterium

The effect of co-culturing a methanogen isolated from a paper mill waste (PMW) with cellulolytic bacteria isolated from the intestinal fluids of the silver cricket (Lepisma saccharina) on the biomethanation of filter paper strips was examined. The autoclaved filter paper strips were subjected to biomethanation in AC 21 medium inoculated with methanogen PMW in the presence and in absence of a co-culture of cellulolytic bacteria. In spite of poor initial response, methane production in the presence of the cellulolytic co-culture were found to increase gradually upto 25 days, after which a reduction in methane production was observed. Analysis of the results in terms of increased cellulose degradation in the presence of cellulolytic bacteria has been made. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bacillus and biopolymer: Prospects and challenges

The microbially derived polyhydroxyalkanoates biopolymers could impact the global climate scenario by replacing the conventional non-degradable, petrochemical-based polymer. The biogenesis, characterization and properties of PHAs by Bacillus species using renewable substrates have been elaborated by many for their wide applications. On the other hand Bacillus species are advantageous over other bacteria due to their abundance even in extreme ecological conditions, higher growth rates even on cheap substrates, higher PHAs production ability, and the ease of extracting the PHAs. Bacillus species possess hydrolytic enzymes that can be exploited for economical PHAs production. This review summarizes the recent trends in both non-growth and growth associated PHAs production by Bacillus species which may provide direction leading to future research towards this growing quest for biodegradable plastics, one more critical step ahead towards sustainable development.     

Sexual dimorphism and polymorphism in a Callovian Phlycticeras (Ammonoidea) assemblage of Kutch, India

An assemblage of Phlycticeras Hyatt from a precisely dated zone of Middle Callovian in Kutch, India has been analyzed. Systematic study reveals that this sample can be divided into two size groups. The larger set shows different adult modifications leading to ornamental polymorphism. Polymorphs are very similar, if not identical, to different chronospecies of Europe, which range between Middle to Upper Callovian. They are grouped under well-known Phlycticeras polygonium (Zieten). The group of smaller specimens on the other hand, strongly resembles a species, which has been previously described as Phlycticeras schaumburgi (Waagen) from the younger Upper Callovian horizons in Kutch. It has a peculiar ‘rooster’-like septicarinate keel in the venter near adult aperture. Phlycticeras-Oecoptychius have been considered as a possible dimorphic pair since long, but here shown to have many inconsistencies to support these views. Instead, sexual dimorphism is explored within Phlycticeras and P. polygonium-P. schaumburgi is proposed as possible antidimorphs. A similar association is found in different stratigraphic assemblages of Europe. Interestingly these two morphs show parallel evolutionary changes within the Phlycticeras genus.