Biogenesis of the Protein Import Channel Tom40 of the Mitochondrial Outer Membrane

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142

Tom40 is the channel-forming subunit of the translocase of the mitochondrial outer membrane (TOM complex), essential for protein import into mitochondria. Tom40 is synthesized in the cytosol and contains information for its mitochondrial targeting and assembly. A number of stable import intermediates have been identified for Tom40 precursors in fungi, the first being an association with the sorting and assembly machinery (SAM) of the outer membrane. By examining the import pathway of human Tom40, we have been able to elucidate additional features in its import. We identify that Hsp90 is involved in delivery of the Tom40 precursor to mitochondria in an ATP-dependent manner. The precursor then forms its first stable intermediate with the outer face of the TOM complex before its membrane integration and assembly. Deletion of an evolutionary conserved region within Tom40 disrupts the TOM complex intermediate and causes it to stall at a new complex in the intermembrane space that we identify to be the mammalian SAM. Unlike its fungal counterparts, the human Tom40 precursor is not found stably arrested at a SAM intermediate. Nevertheless, we show that Tom40 assembly is reduced in mitochondria depleted of human Sam50. These findings are discussed in context with current models from fungal studies.

mentioning

confidence: 70%

Dissection of the Mitochondrial Import and Assembly Pathway for Human Tom40

Humphries

Streimann

Stojanovski

et al. 2005

171

142

“…35 S-labeled Tom40 precursor was imported into isolated mitochondria of the indicated yeast strains. After accumulation of the assembly intermediate I, the mitochondria were reisolated and the Tom40 precursor was chased for an additional 80 min at 25°C (25). The mitochondria were lysed in digitonin and analyzed by blue native electrophoresis and digital autoradiography.…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial Protein Sorting

Meisinger

Wiedemann

Rissler

et al. 2006

Self Cite

The mitochondrial outer membrane contains two distinct machineries for protein import and protein sorting that function in a sequential manner: the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex), which is dedicated to ␤-barrel proteins. The SAM core complex consists of three subunits, Sam35, Sam37, and Sam50, that can associate with a fourth subunit, the morphology component Mdm10, to form the SAM holo complex. Whereas the SAM core complex is required for the biogenesis of all ␤-barrel proteins, Mdm10 and the SAM holo complex play a selective role in ␤-barrel biogenesis by promoting assembly of Tom40 but not of porin. We report that Tom7, a conserved subunit of the TOM complex, functions in an antagonistic manner to Mdm10 in biogenesis of Tom40 and porin. We show that Tom7 promotes segregation of Mdm10 from the SAM holo complex into a low molecular mass form. Upon deletion of Tom7, the fraction of Mdm10 in the SAM holo complex is significantly increased, explaining the opposing functions of Tom7 and Mdm10 in ␤-barrel sorting. Thus the role of Tom7 is not limited to the TOM complex. Tom7 functions in mitochondrial protein biogenesis by a new mechanism, segregation of a sorting component, leading to a differentiation of ␤-barrel assembly.Mitochondria import the vast majority of their ϳ1,000 different proteins from the cytosol (1-3). Upon initial recognition and translocation by the general translocase of the outer mitochondrial membrane (TOM 5 complex), different classes of precursor proteins are distributed to the four mitochondrial subcompartments, outer membrane, intermembrane space, inner membrane, and matrix (4 -11). Preproteins carrying cleavable amino-terminal targeting signals (presequences) are transferred from the TOM complex to the presequence translocase of the inner membrane (TIM23 complex) and then sorted to matrix and inner membrane. Many mitochondrial proteins, however, are synthesized without cleavable presequences, including all outer membrane proteins and the majority of intermembrane space and inner membrane proteins. These proteins use distinct import and sorting machineries. Thus, in addition to the presequence pathway, three sorting pathways for non-cleavable mitochondrial precursor proteins have been identified, each of them starting at the TOM complex. Many hydrophobic inner membrane proteins, including the abundant metabolite carriers, are imported via small Tim proteins of the intermembrane space and the twin pore translocase of the inner membrane (TIM22 complex) (8, 12). Small proteins of the intermembrane space use a special import and assembly machinery that includes Mia40 and the sulfhydryl oxidase Erv1 (13-17). Outer membrane proteins are also initially transported via the TOM complex; however, the TOM complex is not able to integrate ␤-barrel proteins into the membrane. Therefore, the outer membrane contains a separate sorting and assembly machinery (SAM complex) that directs membrane integration and assembly of ␤-barrel...

“…Tom40 forms the translocation channel across the outer membrane and is essential for cell viability under all growth conditions (21,22). Our notion was to slow down mitochondrial protein import by limiting the function of the Tom complex with these mutations.…”

Section: Slowing Down Translocation Allows Cleavage Of Fumarase Bymentioning

confidence: 99%

Translation-coupled Translocation of Yeast Fumarase into Mitochondria in Vivo

Yogev

Karniely

Pines

2007

Fumarase represents proteins that cannot be imported into mitochondria after the termination of translation (post-translationally). Utilizing mitochondrial and cytosolic versions of the tobacco etch virus (TEV) protease, we show that mitochondrially targeted fumarase harboring a TEV protease recognition sequence is efficiently cleaved by the mitochondrial but not by the cytosolic TEV protease. Nonetheless, fumarase was readily cleaved by cytosolic TEV when its import into mitochondria was slowed down by either (i) disrupting the activity of the TOM complex, (ii) lowering the growth temperature, or (iii) reducing the inner membrane electrochemical potential. Accessibility of the fumarase nascent chain to TEV protease under such conditions was prevented by low cycloheximide concentrations, which impede translation. In addition, depletion of the ribosome-associated nascent polypeptide-associated complex (NAC) reduced the fumarase rate of translocation into mitochondria and exposed it to TEV cleavage in the cytosol. These results indicate that cytosolic exposure of the fumarase nascent chain depends on both translocation and translation rates, allowing us to discuss the possibility that import of fumarase into mitochondria occurs while the ribosome is still attached to the nascent chain.