Rupture of the mitochondrial outer membrane impairs porin assembly.

148

134

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro–imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

Section: Discussionmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Biogenesis of Porin of the Outer Mitochondrial Membrane Involves an Import Pathway via Receptors and the General Import Pore of the Tom Complex

Krimmer

Rapaport

Ryan

et al. 2001

148

134

“…9 A; Hwang et al, 1989;Rapaport and Neupert, 1999;Krimmer et al, 2001;Stan et al, 2003). As was observed before, rupturing the outer membrane resulted in substantial reduction in the insertion effi ciency of porin precursor (Smith et al, 1994). This reduction is probably caused by the loss of the small Tim proteins, which were shown to be involved in the biogenesis of β-barrel proteins (Hoppins and Nargang, 2004;Wiedemann et al, 2004;Habib et al, 2005).…”

Section: Translocation Of Porin Precursor Across the Tom Complex Is Required For Its Effi Cient Insertion Into The Outer Membranementioning

confidence: 85%

The N-terminal domain of Tob55 has a receptor-like function in the biogenesis of mitochondrial β-barrel proteins

Habib

Waizenegger

Niewienda

et al. 2006

β-Barrel proteins constitute a distinct class of mitochondrial outer membrane proteins. For import into mitochondria, their precursor forms engage the TOM complex. They are then relayed to the TOB complex, which mediates their insertion into the outer membrane. We studied the structure–function relationships of the core component of the TOB complex, Tob55. Tob55 precursors with deletions in the N-terminal domain were not affected in their targeting to and insertion into the mitochondrial outer membrane. Replacement of wild-type Tob55 by these deletion variants resulted in reduced growth of cells, and mitochondria isolated from such cells were impaired in their capacity to import β-barrel precursors. The purified N-terminal domain was able to bind β-barrel precursors in a specific manner. Collectively, these results demonstrate that the N-terminal domain of Tob55 recognizes precursors of β-barrel proteins. This recognition may contribute to the coupling of the translocation of β-barrel precursors across the TOM complex to their interaction with the TOB complex.

“…However, in normal physiology, other considerations apply: the question of membrane specificity; the problem of competing interactions of VDAC with other proteins, specific or otherwise; the necessity to maintain a conformation of soluble VDAC after release from the ribosome that is compatible with subsequent membrane insertion; and the requirement for a mechanism to catalyze VDAC insertion upon contact with the mitochondrial outer membrane. In the import reaction documented here, the appropriate translocation-competent conformation for VDAC may be supplied by chaperones/factors present in reticulocyte lysate (Smith et al, 1994). The only other minimum requirement was provided by hTom20 on the surface of the membrane.…”

Section: Resultsmentioning

confidence: 99%

Direct Membrane Insertion of Voltage-dependent Anion-selective Channel Protein Catalyzed by Mitochondrial Tom20

Schleiff

Silvius

Shore

1999

Insertion of newly synthesized proteins into or across the mitochondrial outer membrane is initiated by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into a preprotein translocation pore, comprised of Tom40. Here, we show that a prominent β-barrel channel protein spanning the outer membrane, human voltage- dependent anion-selective channel (VDAC), bypasses the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP. Therefore, Tom20 transforms newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer.