scholarly journals Mitochondrial and nonmitochondrial citrate synthases in Saccharomyces cerevisiae are encoded by distinct homologous genes.

1986 ◽  
Vol 6 (12) ◽  
pp. 4509-4515 ◽  
Author(s):  
M Rosenkrantz ◽  
T Alam ◽  
K S Kim ◽  
B J Clark ◽  
P A Srere ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Citrate Synthase ◽  
Amino Terminus ◽  
Translation Product ◽  
Mitochondrial Targeting ◽  
E Coli ◽  
Homologous Genes ◽  
Primary Translation Product ◽  
Mitochondrial Targeting Sequences

Saccharomyces cerevisiae contains two genes, CIT1 and CIT2, encoding functional citrate synthase (K.-S. Kim, M. S. Rosenkrantz, and L. Guarente, Mol. Cell. Biol. 6:1936-1942, 1986). We show here that CIT2 encodes a nonmitochondrial form of citrate synthase. The DNA sequence of CIT2 presented provides a possible explanation for why the CIT2 product, unlike the CIT1 product, fails to be imported into mitochondria. While the products of these two genes are highly homologous, they diverge strikingly at their amino termini. The amino terminus of the CIT1 primary translation product extends 39 residues beyond the amino termini of Escherichia coli and porcine citrate synthases. This extension consists of a typical mitochondrial targeting motif. The amino terminus of the CIT2 primary translation product extends 20 residues beyond the amino termini of the E. coli and porcine enzymes. The CIT2-encoded extension is not homologous to that of CIT1, resulting in a nonmitochondrial localization of the product. The CIT2-encoded extension, however, does bear certain similarities to mitochondrial targeting sequences. The possible role of this sequence in targeting this CIT2 product to a nonmitochondrial organelle is discussed.

Mitochondrial and nonmitochondrial citrate synthases in Saccharomyces cerevisiae are encoded by distinct homologous genes

1986 ◽  
Vol 6 (12) ◽  
pp. 4509-4515
Author(s):  
M Rosenkrantz ◽  
T Alam ◽  
K S Kim ◽  
B J Clark ◽  
P A Srere ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Citrate Synthase ◽  
Amino Terminus ◽  
Translation Product ◽  
Mitochondrial Targeting ◽  
E Coli ◽  
Homologous Genes ◽  
Primary Translation Product ◽  
Mitochondrial Targeting Sequences

Saccharomyces cerevisiae contains two genes, CIT1 and CIT2, encoding functional citrate synthase (K.-S. Kim, M. S. Rosenkrantz, and L. Guarente, Mol. Cell. Biol. 6:1936-1942, 1986). We show here that CIT2 encodes a nonmitochondrial form of citrate synthase. The DNA sequence of CIT2 presented provides a possible explanation for why the CIT2 product, unlike the CIT1 product, fails to be imported into mitochondria. While the products of these two genes are highly homologous, they diverge strikingly at their amino termini. The amino terminus of the CIT1 primary translation product extends 39 residues beyond the amino termini of Escherichia coli and porcine citrate synthases. This extension consists of a typical mitochondrial targeting motif. The amino terminus of the CIT2 primary translation product extends 20 residues beyond the amino termini of the E. coli and porcine enzymes. The CIT2-encoded extension is not homologous to that of CIT1, resulting in a nonmitochondrial localization of the product. The CIT2-encoded extension, however, does bear certain similarities to mitochondrial targeting sequences. The possible role of this sequence in targeting this CIT2 product to a nonmitochondrial organelle is discussed.

scholarly journals Proteolytic Processing of a Serotype 8 Human Astrovirus ORF2 Polyprotein

2002 ◽  
Vol 76 (16) ◽  
pp. 7996-8002 ◽  
Author(s):  
Ernesto Méndez ◽  
Teresa Fernández-Luna ◽  
Susana López ◽  
Martha Méndez-Toss ◽  
Carlos F. Arias
Keyword(s):  
Host Cell ◽  
Immunoblot Analysis ◽  
Proteolytic Processing ◽  
Amino Terminus ◽  
Translation Product ◽  
Carboxy Terminus ◽  
Trypsin Treatment ◽  
Primary Translation Product ◽  
Viral Particles ◽  
Human Astrovirus

ABSTRACT Astroviruses require the proteolytic cleavage of the capsid protein to infect the host cell. Here we describe the processing pathway of the primary translation product of the structural polyprotein (ORF2) encoded by a human astrovirus serotype 8 (strain Yuc8). The primary translation product of ORF2 is of approximately 90 kDa, which is subsequently cleaved to yield a 70-kDa protein (VP70) which is assembled into the viral particles. Limited trypsin treatment of purified particles containing VP70 results in the generation of polypeptides VP41 and VP28, which are then further processed to proteins of 38.5, 35, and 34 kDa and 27, 26, and 25 kDa, respectively. VP34, VP27 and VP25 are the predominant proteins in fully cleaved virions, which correlate with the highest level of infectivity. Processing of the VP41 protein to yield VP38.5 to VP34 polypeptides occurred at its carboxy terminus, as suggested by immunoblot analysis using hyperimmune sera to different regions of the ORF2, while processing of VP28 to generate VP27 and VP25 occurred at its carboxy and amino terminus, respectively, as determined by immunoblot, as well as by N-terminal sequencing of those products. Based on these data, the processing pathway for the 90-kDa primary product of astrovirus Yuc8 ORF2 is presented.

scholarly journals The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis

1999 ◽  
Vol 338 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Evelyne RAUX ◽  
Treasa McVEIGH ◽  
Sarah E. PETERS ◽  
Thomas LEUSTEK ◽  
Martin J. WARREN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Biosynthetic Pathways ◽  
Subtle Difference ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
His Tag ◽  
Cobalamin Biosynthesis

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.

scholarly journals PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis.

1991 ◽  
Vol 114 (6) ◽  
pp. 1167-1178 ◽  
Author(s):  
J Höhfeld ◽  
M Veenhuis ◽  
W H Kunau
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Amino Terminus ◽  
Peroxisome Biogenesis ◽  
Coding Region ◽  
Gene Encoding ◽  
Peroxisomal Membrane ◽  
E Coli ◽  
Protease Digestion

Saccharomyces cerevisiae pas3-mutants are described which conform the pas-phenotype recently reported for the peroxisomal assembly mutants pas1-1 and pas2 (Erdmann, R., M. Veenhuis, D. Mertens, and W.-H Kunau, 1989, Proc. Natl. Acad. Sci. USA. 86:5419-5423). The isolation of pas3-mutants enabled us to clone the PAS3 gene by functional complementation. DNA sequence analysis revealed a 50.6-kD protein with at least one domain of sufficient length and hydrophobicity to span a lipid bilayer. To verify these predictions antibodies were raised against a truncated portion of the PAS3 coding region overexpressed in E. coli. Pas3p was identified as a 48 kD peroxisomal integral membrane protein. It is shown that a lack of this protein causes the peroxisome-deficient phenotype and the cytosolic mislocalization of peroxisomal matrix enzymes. Based on protease digestion experiments Pas3p is discussed to be anchored in the peroxisomal membrane by its amino-terminus while the bulk of the molecule is exposed to the cytosol. These findings are consistent with the possibility that Pas3p is one component of the peroxisomal import machinery.

scholarly journals Expression of the Mitochondrial ADP/ATP Carrier inEscherichia coli

2001 ◽  
Vol 276 (15) ◽  
pp. 11499-11506 ◽  
Author(s):  
Simone Heimpel ◽  
Gabriele Basset ◽  
Sabine Odoy ◽  
Martin Klingenberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Inclusion Bodies ◽  
Positive Charge ◽  
Inescherichia Coli ◽  
Transport Activity ◽  
E Coli ◽  
The Matrix ◽  
A Charge

Previously, the role of residues in the ADP/ATP carrier (AAC) fromSaccharomyces cerevisiaehas been studied by mutagenesis, but the dependence of mitochondrial biogenesis on functional AAC impedes segregation of the mutational effects on transport and biogenesis. Unlike other mitochondrial carriers, expression of the AAC from yeast or mammalians inEscherichia coliencountered difficulties because of disparate codon usage. Here we introduce the AAC fromNeurospora crassainE. coli, where it is accumulated in inclusion bodies and establish the reconstitution conditions. AAC expressed with heat shock vector gave higher activity than with pET-3a. Transport activity was absolutely dependent on cardiolipin. The 10 single mutations of intrahelical positive residues and of the matrix repeat (+X+) motif resulted in lower activity, except of R245A. R143A had decreased sensitivity toward carboxyatractylate. The ATP-linked exchange is generally more affected than ADP exchange. This reflects a charge network that propagates positive charge defects to ATP4−more strongly than to ADP3−transport. Comparison to the homologous mutants of yeast AAC2 permits attribution of the roles of these residues more to ADP/ATP transport or to AAC import into mitochondria.

scholarly journals Role of PITRM1 in Mitochondrial Dysfunction and Neurodegeneration

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 833
Author(s):  
Dario Brunetti ◽  
Alessia Catania ◽  
Carlo Viscomi ◽  
Michela Deleidi ◽  
Laurence A. Bindoff ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Mitochondrial Fraction ◽  
Current Evidence ◽  
Mitochondrial Targeting ◽  
Therapeutic Approaches ◽  
Atp Production ◽  
Precursor Proteins ◽  
Toxic Peptides ◽  
Mitochondrial Targeting Sequences

Mounting evidence shows a link between mitochondrial dysfunction and neurodegenerative disorders, including Alzheimer Disease. Increased oxidative stress, defective mitodynamics, and impaired oxidative phosphorylation leading to decreased ATP production, can determine synaptic dysfunction, apoptosis, and neurodegeneration. Furthermore, mitochondrial proteostasis and the protease-mediated quality control system, carrying out degradation of potentially toxic peptides and misfolded or damaged proteins inside mitochondria, are emerging as potential pathogenetic mechanisms. The enzyme pitrilysin metallopeptidase 1 (PITRM1) is a key player in these processes; it is responsible for degrading mitochondrial targeting sequences that are cleaved off from the imported precursor proteins and for digesting a mitochondrial fraction of amyloid beta (Aβ). In this review, we present current evidence obtained from patients with PITRM1 mutations, as well as the different cellular and animal models of PITRM1 deficiency, which points toward PITRM1 as a possible driving factor of several neurodegenerative conditions. Finally, we point out the prospect of new diagnostic and therapeutic approaches.

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae

1992 ◽  
Vol 12 (12) ◽  
pp. 5593-5599
Author(s):  
K K Singh ◽  
G M Small ◽  
A S Lewin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Citrate Synthase ◽  
Signal Sequence ◽  
Carboxyl Terminus ◽  
Density Gradients ◽  
Mitochondrial Targeting ◽  
Amino Terminal ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.

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