Negatively charged amino acid residues play an active role in orienting the Sec-independent Pf3 coat protein in the<i>Escherichia coli</i>inner membrane

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N-and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for secindependent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.

Section: Discussioncontrasting

confidence: 53%

N-terminal Tail Export from the Mitochondrial Matrix

Rojo

Guiard²,

Neupert

et al. 1999

“…the first step is targeting of Pf3 coat protein to the membrane, the second step is partitioning of the Pf3 coat hydrophobic domain into the membrane lipid bilayer, and the final step is the formation of a transmembrane helix with concomitant membrane translocation of the amino-terminal region (23). In the third step, the proton motive force (pmf) is required for the electrophoretic transfer of the amino-terminal tail, which contains two negatively charged amino acid residues (33). Our data obtained by photocrosslinking suggests that YidC functions at the stage of membrane insertion, which might be the membrane partitioning step (the second step) or the orientation step (the third step), to form the transmembrane form of the protein.…”

Section: Yidcmentioning

confidence: 99%

Direct Interaction of YidC with the Sec-independent Pf3 Coat Protein during Its Membrane Protein Insertion

Chen¹,

Samuelson²,

Jiang³

et al. 2002

Self Cite

128

120

YidC is a newly defined translocase component that mediates the insertion of proteins into the membrane bilayer. How YidC functions in the insertion process is not known. In this study, we report that the Sec-independent Pf3 coat protein requires the YidC protein specifically for the membrane translocation step. Using photocrosslinking techniques and ribosome-bound Pf3 coat derivatives with an extended carboxyl-terminal region, we found that the transmembrane region of the Pf3 coat protein physically interacts with YidC and the bacterial signal recognition particle Ffh component. We also find that in the insertion pathway, Pf3 coat interacts strongly with YidC only after its transmembrane segment is fully exposed outside the ribosome tunnel. Interaction between Pf3 coat and YidC occurs even in the absence of the proton motive force and with a Pf3 coat mutant that is defective in transmembrane insertion. Our study demonstrates that YidC can directly interact with a Sec-independent membrane protein, and the role of YidC is at the stage of folding the Pf3 protein into a transmembrane configuration.

“…This export process resembles the bacterial N-tail export mechanism of the M13 procoat protein; it depends on the membrane potential and an Oxa1p homologue, YidC (17,27). The N terminus of Pf3 coat protein is exported in a membrane potential-dependent manner (25). Therefore, we examined whether the export of the N terminus of T7-Su8 depends on the membrane potential and/or ATP.…”

Section: Insertion Of T7-su8 Across the Membrane Depends On Thementioning

confidence: 99%

“…The M13 procoat protein is synthesized with a cleavable signal peptide and spans the membrane once with the processed N terminus exposed to the periplasm and the C terminus remaining in the cytoplasm (18 -24). The Pf3 coat protein is synthesized without a cleavable signal sequence and inserts into the membrane once, exposing the N-terminal segment to the periplasm and the C-terminal segment to the cytoplasm (25,26). In contrast to the predicted direct insertion mechanism, Samuelson et al (27) demonstrated that insertion of the M13 procoat protein across the membrane depends on a Oxa1p homologue, YidC.…”

mentioning

confidence: 99%

Insertion of Mitochondrial DNA-encoded F1F0-ATPase Subunit 8 across the Mitochondrial Inner Membrane in Vitro

Miki

Mihara

2001

Cytochrome oxidase subunits I, II, and III, the mitochondrial DNA-encoded proteins, are inserted across the inner membrane by the Oxa1p-containing translocator in a membrane potential-dependent manner. Oxa1p is also involved in the insertion of the cytoplasmically synthesized precursor of Oxa1p itself into the inner membrane from the matrix via the conservative sorting pathway. The mechanism of insertion of the other mitochondrially synthesized proteins, however, is unexplored. The insertion of the mitochondrial DNA-encoded subunit 8 of F 1 F 0 -ATPase (Su8) across the inner membrane was analyzed in vitro using the inverted inner membrane vesicles and the Escherichia coli lysate-synthesized substrate. This assay revealed that the N-terminal segment of Su8 inserted across the membrane to the intermembrane space and assumed the correct trans-cis topology depending on the mitochondrial matrix fraction. This translocation reaction was similar to those of Sec-independent, direct insertion pathways of E. coli and chloroplast thylakoid membranes. (i) It required neither nucleotide triphosphates nor membrane potential, and hydrophobic forces drove the process. (ii) It did not require protease-sensitive membrane components facing the matrix space. (iii) It could be inserted across liposomes in the correct topology in a matrix fraction-dependent manner. Thus, a novel mechanism conserved in bacteria and chloroplasts also functions in the insertion of Su8 across the mitochondrial inner membrane.