The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism.

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: Discussionmentioning

confidence: 81%

N-terminal Tail Export from the Mitochondrial Matrix

Rojo

Guiard²,

Neupert

et al. 1999

“…We investigated whether a mutant Procoat-Lep with a net charge of zero in the periplasmic loop (0PClep) still requires YidC for insertion. 0PCLep has been shown previously to insert independently of an electrochemical potential (26). Fig.…”

Section: Yidc Functions To Promote Membrane Insertion Of Both Membranmentioning

confidence: 79%

“…Therefore, we investigated whether YidC, the bacterial Oxa1p homologue, can promote insertion of Procoat proteins that insert independently of the membrane potential. Mutants of Procoat-Lep have been studied extensively to examine the importance of the membrane electrochemical potential in membrane insertion (26). The extension of the Procoat with the Lep P2 domain allowed us to immunoprecipitate a wide variety of Procoat mutants, which are not recognized by the Procoat antisera.…”

Section: Yidc Functions To Promote Membrane Insertion Of Both Membranmentioning

confidence: 99%

See 1 more Smart Citation

Function of YidC for the Insertion of M13 Procoat Protein inEscherichia coli

Samuelson¹,

Jiang²,

Liang³

et al. 2001

127

The membrane insertion of the Sec-independent M13 Procoat protein in bacteria requires the membrane electrochemical potential and the integral membrane protein YidC. We show here that YidC is involved in the translocation but not in the targeting of the Procoat protein, because we found the protein was partitioned into the membrane in the absence of YidC. YidC can function also to promote membrane insertion of Procoat mutants that insert independently of the membrane potential, proving that the effect of YidC depletion is not due to a dissipation of the membrane potential. We also found that YidC is absolutely required for Sec-dependent translocation of a long periplasmic loop of a mutant Procoat in which the periplasmic region has been extended from 20 to 194 residues. Furthermore, when Secdependent membrane proteins with large periplasmic domains were overproduced under YidC-limited conditions, we found that the exported proteins pro-OmpA and pre-peptidoglycan-associated lipoprotein accumulated in the cytoplasm. This suggests for Sec-dependent proteins that YidC functions at a late stage in membrane insertion, after the Sec translocase interacts with the translocating membrane protein. These studies are consistent with the understanding that YidC cooperates with the Sec translocase for membrane translocation and that YidC is required for clearing the protein-conducting channel.

“…The Proton Motive Force Is Required for Membrane Insertion of FtsQ-The PMF has been shown to play an important role in the insertion of some IMPs (27) and membrane topology control (28,29). The ATP needed for the translation reaction is also used by the F 1 F 0 ATPase to generate a PMF.…”

Section: Co-translational Insertion Of Ftsq Intomentioning

confidence: 99%

SecYEG Proteoliposomes Catalyze the Δϕ-Dependent Membrane Insertion of FtsQ

Laan

Nouwen

Driessen

2004

In Escherichia coli, the insertion of most inner membrane proteins is mediated by the Sec translocase. Ribosome-bound nascent chains of Sec-dependent inner membrane proteins are targeted to the SecYEG complex via the signal recognition particle pathway. We now demonstrate that the signal recognition particledependent co-translational membrane targeting and membrane insertion of FtsQ can be reconstituted with proteoliposomes containing purified SecYEG. SecA and a transmembrane electrical potential are essential for the translocation of the large periplasmic domain of FtsQ, whereas co-reconstituted YidC has an inhibitory effect. These data demonstrate that membrane protein insertion can be reconstituted with a minimal set of purified Sec components.