Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency

Galanis, Maria; Devenish, Rodney J.; Nagley, Phillip

doi:10.1016/0014-5793(91)80529-c

Cited by 51 publications

(26 citation statements)

References 20 publications

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“…The duplication of the MTS was found to improve the in vitro and in vivo import of hydrophobic proteins into yeast mitochondria (32,39). It was suggested that a long MTS can improve the interaction of the precursor with the mitochondrial import machinery.…”

Section: Discussionmentioning

confidence: 99%

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The Typically Mitochondrial DNA-encoded ATP6 Subunit of the F1F0-ATPase Is Encoded by a Nuclear Gene in Chlamydomonas reinhardtii

Funes¹,

Davidson²,

Claros³

et al. 2002

Journal of Biological Chemistry

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The atp6 gene, encoding the ATP6 subunit of F 1 F 0 -ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomonas reinhardtii. Analysis of C. reinhardtii expressed sequence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5-and 3-RACE were used to obtain the complete cDNA and genomic sequences of C. reinhardtii atp6. The atp6 gene exhibited characteristics of a nucleus-encoded gene: Southern hybridization signals consistent with nuclear localization, the presence of introns, and a codon usage and a polyadenylation signal typical of nuclear genes. The corresponding ATP6 protein was confirmed as a subunit of the mitochondrial F 1 F 0 -ATP synthase from C. reinhardtii by N-terminal sequencing. The predicted ATP6 polypeptide has a 107-amino acid cleavable mitochondrial targeting sequence. The mean hydrophobicity of the protein is decreased in those transmembrane regions that are predicted not to participate directly in proton translocation or in intersubunit contacts with the multimeric ring of c subunits. This is the first example of a mitochondrial protein with more than two transmembrane stretches, directly involved in proton translocation, that is nucleus-encoded.

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Section: Discussionmentioning

confidence: 99%

“…The hypothetical boundary between importable and nonimportable proteins, indicated by a broad, gray diagonal, was derived from Claros et al (39) and Pérez-Martínez et al (11). The following proteins with their symbols were analyzed:…”

Section: Discussionmentioning

confidence: 99%

The Typically Mitochondrial DNA-encoded ATP6 Subunit of the F1F0-ATPase Is Encoded by a Nuclear Gene in Chlamydomonas reinhardtii

Funes¹,

Davidson²,

Claros³

et al. 2002

Journal of Biological Chemistry

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“…These multiple Tom20-recognition motifs could offer a second C-terminal Tom20-binding element, like the pSu9 presequence, to increase the import efficiencies. In this connection, it would be interesting to note that the presequence with a duplication of pSu9 further improved the import efficiency of mitochondrial precursor proteins (22). In order to further confirm that a second Tom20 binding element is not specific to the Su9 presequence, we picked up the presequence of Mprl3, a mitochondrial ribosomal protein of the large subunit 1, as one of the long presequences that was predicted to have second Tom20-binding elements.…”

Section: Discussionmentioning

confidence: 99%

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria

Yamamoto

Itoh

Kawano

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.protein's function relies on its correct subcellular location. Newly synthesized proteins are delivered to their sites of actions by cellular protein transport systems. Because subcellular compartments are bounded by biological membrane(s) in eukaryotic cells, the protein transport needs to start with insertion into or translocation across the target membrane. Therefore most protein transport systems involve a targeting signal on the cargo protein and a multiprotein complex on the target membrane called a translocator (1).Mitochondria are essential organelles in eukaryotic cells that consist of the outer and inner membranes and two aqueous compartments, the intermembrane space (IMS) and the matrix. Most mitochondrial proteins are encoded in the nuclear genome, synthesized in the cytosol, and subsequently imported into mitochondria. Mitochondrial protein import is mediated by translocators in the outer and inner membranes, including two TOM (translocase of the outer mitochondrial membrane) complexes, the TOM40 complex and the TOB (topogenesis of outer-membrane β-barrel proteins)/SAM (sorting and assembly machinery) complex, and two TIM (translocase of the inner mitochondrial membrane) complexes, the TIM23 complex and the TIM22 complex (2-5). The import pathway generally starts from the TOM40 complex and then branches out into several distinct intramitochondrial sorting pathways involving other translocators.The targeting information for mitochondria is contained in the N-terminal cleavable presequence or within the mature part of precursor proteins. The targeting information as well as intramitochondrial sorting information is recognized by several receptor subunits of the TOM and TIM complexes along the import pathways. Among them, Tom20, a peripheral subunit of the TOM40 complex, is a general import receptor that recognizes mitochondrial targeting signals contained in presequences. Tom20 is anchored to the outer membrane by the N-terminal hydrophobic segment and exposes a receptor domain to the cytosol. The NMR and X-ray structures of the receptor domain of rat Tom20 in a complex with a presequence peptide showed that the bound presequence forms an amphiphilic helix...

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“…These results indicated that the N-terminal 13 residues (including the first Val) are essential for the biosynthesis of nosiheptide. Previous studies have shown that a tandem duplication of the mitochondrion-targeting leader sequence is capable of improving the efficiency of delivery of the protein into mitochondrial matrix (23). If the leader peptide was a target region for PTM enzymes, a tandem duplicated leader sequence would enhance the interaction between the precursor and PTM enzymes, which might be favorable to the posttranslational processing to produce nosiheptide.…”

Section: Resultsmentioning

confidence: 99%

Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

Jin

Xue

et al. 2017

Appl Environ Microbiol

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Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrugresistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N-and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs.IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the biosynthetic process, the instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides.

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Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency

Cited by 51 publications

References 20 publications

The Typically Mitochondrial DNA-encoded ATP6 Subunit of the F1F0-ATPase Is Encoded by a Nuclear Gene in Chlamydomonas reinhardtii

The Typically Mitochondrial DNA-encoded ATP6 Subunit of the F1F0-ATPase Is Encoded by a Nuclear Gene in Chlamydomonas reinhardtii

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria

Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

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