scholarly journals Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide.

1991 ◽  
Vol 113 (1) ◽  
pp. 65-76 ◽  
Author(s):  
G Isaya ◽  
F Kalousek ◽  
W A Fenton ◽  
L E Rosenberg
Keyword(s):  
Single Step ◽  
Mitochondrial Proteins ◽  
Leader Peptide ◽  
Amino Terminus ◽  
Mature Protein ◽  
Amino Terminal ◽  
Protease Cleavage ◽  
Iron Sulfur ◽  
Mitochondrial Processing Peptidase ◽  
Processing Peptidase

Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re-inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility.

scholarly journals Stromal Processing Peptidase Binds Transit Peptides and Initiates Their Atp-Dependent Turnover in Chloroplasts

1999 ◽  
Vol 147 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Stefan Richter ◽  
Gayle K. Lamppa
Keyword(s):  
Binding Assay ◽  
Specific Binding ◽  
Transit Peptide ◽  
Binding Motif ◽  
Mature Protein ◽  
Sequence Of Events ◽  
Mitochondrial Processing Peptidase ◽  
Transit Peptides ◽  
Processing Peptidase ◽  
Zinc Binding Motif

A stromal processing peptidase (SPP) cleaves a broad range of precursors targeted to the chloroplast, yielding proteins for numerous biosynthetic pathways in different compartments. SPP contains a signature zinc-binding motif, His-X-X-Glu-His, that places it in a metallopeptidase family which includes the mitochondrial processing peptidase. Here, we have investigated the mechanism of cleavage by SPP, a late, yet key event in the import pathway. Recombinant SPP removed the transit peptide from a variety of precursors in a single endoproteolytic step. Whereas the mature protein was immediately released, the transit peptide remained bound to SPP. SPP converted the transit peptide to a subfragment form that it no longer recognized. We conclude that SPP contains a specific binding site for the transit peptide and additional proteolysis by SPP triggers its release. A stable interaction between SPP and an intact transit peptide was directly demonstrated using a newly developed binding assay. Unlike recombinant SPP, a chloroplast extract rapidly degraded both the transit peptide and subfragment. A new degradative activity, distinguishable from SPP, was identified that is ATP- and metal-dependent. Our results indicate a regulated sequence of events as SPP functions during precursor import, and demonstrate a previously unrecognized ATP-requirement for transit peptide turnover.

scholarly journals The Monothiol Single-Domain Glutaredoxin Is Conserved in the Highly Reduced Mitochondria of Giardia intestinalis

2009 ◽  
Vol 8 (10) ◽  
pp. 1584-1591 ◽  
Author(s):  
Petr Rada ◽  
Ondřej Šmíd ◽  
Robert Sutak ◽  
Pavel Doležal ◽  
Jan Pyrih ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Immunofluorescence Microscopy ◽  
Giardia Intestinalis ◽  
Cluster Assembly ◽  
Mature Protein ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Processing Peptidase ◽  
Mitochondrial Origin ◽  
Active Site Sequence

ABSTRACT The highly reduced mitochondria (mitosomes) of Giardia intestinalis are recently discovered organelles for which, it was suggested, iron-sulfur cluster assembly was their only conserved function. However, only an incomplete set of the components required for FeS cluster biogenesis was localized to the mitosomes. Via proteomic analysis of a mitosome-rich cellular fraction together with immunofluorescence microscopy, we identified a novel mitosomal protein homologous to monothiol glutaredoxins containing a CGFS motif at the active site. Sequence analysis revealed the presence of long nonconserved N-terminal extension of 77 amino acids, which was absent in the mature protein. Expression of the complete and N-terminally truncated forms of the glutaredoxin indicated that the extension is involved in glutaredoxin import into mitosomes. However, the mechanism of preprotein processing is unclear, as the mitosomal processing peptidase is unable to cleave this type of extension. The recombinant mature protein was shown to form a homodimeric structure, which binds a labile FeS cluster. The cluster is stabilized by glutathione and dithiothreitol. Phylogenetic analysis showed that giardial glutaredoxin is related to the mitochondrial monothiol glutaredoxins involved in FeS cluster assembly. The identification of a mitochondrial-type monothiol glutaredoxin in the mitosomes of G. intestinalis thus completes the mitosomal FeS cluster biosynthetic pathway and provides further evidence for the mitochondrial origin of these organelles.

scholarly journals Homozygous YME1L1 mutation causes mitochondriopathy with optic atrophy and mitochondrial network fragmentation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Bianca Hartmann ◽  
Timothy Wai ◽  
Hao Hu ◽  
Thomas MacVicar ◽  
Luciana Musante ◽  
...  
Keyword(s):  
High Energy ◽  
Mitochondrial Proteins ◽  
Homozygous Mutation ◽  
Mitochondrial Network ◽  
Optic Nerve Atrophy ◽  
Rapid Degradation ◽  
Mitochondrial Processing Peptidase ◽  
Energy Consuming ◽  
Processing Peptidase ◽  
Multisystemic Disorders

Mitochondriopathies often present clinically as multisystemic disorders of primarily high-energy consuming organs. Assembly, turnover, and surveillance of mitochondrial proteins are essential for mitochondrial function and a key task of AAA family members of metalloproteases. We identified a homozygous mutation in the nuclear encoded mitochondrial escape 1-like 1 gene YME1L1, member of the AAA protease family, as a cause of a novel mitochondriopathy in a consanguineous pedigree of Saudi Arabian descent. The homozygous missense mutation, located in a highly conserved region in the mitochondrial pre-sequence, inhibits cleavage of YME1L1 by the mitochondrial processing peptidase, which culminates in the rapid degradation of YME1L1 precursor protein. Impaired YME1L1 function causes a proliferation defect and mitochondrial network fragmentation due to abnormal processing of OPA1. Our results identify mutations in YME1L1 as a cause of a mitochondriopathy with optic nerve atrophy highlighting the importance of YME1L1 for mitochondrial functionality in humans.

Distribution of dihydrofolate reductase-thymidylate synthetase polypeptide in methotrexateresistant and wild type Leishmania major: Evidence for cytoplasmic distribution despite a hydrophilic leader sequence

1990 ◽  
Vol 48 (3) ◽  
pp. 610-611
Author(s):  
J. R. Swafford ◽  
S. M. Beverley ◽  
C. C. Kan ◽  
J. P. Caulfield
Keyword(s):  
Dihydrofolate Reductase ◽  
Leishmania Major ◽  
Thymidylate Synthetase ◽  
Mitochondrial Proteins ◽  
Amino Terminus ◽  
Mature Protein ◽  
Wild Type ◽  
Hydrophilic Amino Acid ◽  
Higher Eukaryotes ◽  
Single Polypeptide

The housekeeping enzymes, dihydrofolate reductase (DHFR) and thymidylate synthetase (TS) are encoded by separate genes and located in the cytoplasm of higher eukaryotes. However, in the trypanosomatid protozoan Leishmania these enzymes are both located on a single polypeptide. The DNA sequence for this polypeptide predicts a 23 hydrophilic amino acid extension from the amino terminus that resembles the import sequence of mitochondrial proteins in other species. It is not known whether the hydrophilic leader actually occurs in the mature protein in Leishmania since the amino terminus is blocked.Here, we have examined the distribution of the DHFR-TS peptide in two strains of Leishmania major in order to see if there was a preferential distribution of the protein in the mitochondrion.

scholarly journals A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

1998 ◽  
Vol 9 (8) ◽  
pp. 2201-2216 ◽  
Author(s):  
Thu Nguyen ◽  
Dani B.N. Vinh ◽  
Douglas K. Crawford ◽  
Trisha N. Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Terminus ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Synthetic Lethal ◽  
Lethal Phenotype ◽  
Amino Terminal ◽  
N Terminus ◽  
Two Hybrid

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations inSPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, thespc97 alleles are synthetic lethal withspc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

scholarly journals Maturation of Mitochondrially Targeted Prx V Involves a Second Cleavage by Mitochondrial Intermediate Peptidase That Is Sensitive to Inhibition by H2O2

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 346
Author(s):  
Juhyun Sim ◽  
Jiyoung Park ◽  
Hyun Ae Woo ◽  
Sue Goo Rhee
Keyword(s):  
Short Form ◽  
Mitochondrial Matrix ◽  
Mitochondrial Processing Peptidase ◽  
Mouse Tissues ◽  
N Terminus ◽  
Mitochondrial Intermediate Peptidase ◽  
Subsequent Degradation ◽  
Processing Peptidase ◽  
Mitochondria Targeting ◽  
Over Time

Prx V mRNA contains two in-frame AUG codons, producing a long (L-Prx V) and short form of Prx V (S-Prx V), and mouse L-Prx V is expressed as a precursor protein containing a 49-amino acid N-terminal mitochondria targeting sequence. Here, we show that the N-terminal 41-residue sequence of L-Prx V is cleaved by mitochondrial processing peptidase (MPP) in the mitochondrial matrix to produce an intermediate Prx V (I-Prx V) with a destabilizing phenylalanine at its N-terminus, and further, that the next 8-residue sequence is cleaved by mitochondrial intermediate peptidase (MIP) to convert I-Prx V to a stabilized mature form that is identical to S-Prx V. Further, we show that when mitochondrial H2O2 levels are increased in HeLa cells using rotenone, in several mouse tissues by deleting Prx III, and in the adrenal gland by deleting Srx or by exposing mice to immobilized stress, I-Prx V accumulates transiently and mature S-Prx V levels decrease in mitochondria over time. These findings support the view that MIP is inhibited by H2O2, resulting in the accumulation and subsequent degradation of I-Prx V, identifying a role for redox mediated regulation of Prx V proteolytic maturation and expression in mitochondria.

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