THE MITOCHONDRIAL GENETIC SYSTEM ....•••••• ••••••••••.• ••••••••••..•......... 291 Organization and Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . 291 Genetic Content 296 Regulation of Mitochondrial Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

Biogenesis of Mitochondria

Mitochondria import many hundreds of different proteins that are encoded by nuclear genes These proteins are targeted to the mitochondria, translocated through the mitochondrial membranes, and sorted to the different mitochondrial subcompartments Separate translocases in the mitochondrial outer membrane (TOM complex) and in the inner membrane (TIM complex) facilitate recognition of preproteins and transport across the two membranes Factors in the cytosol assist in targeting of preproteins Protein components in the matrix partake in energetically driving translocation in a reaction that depends on the membrane potential and matrix-ATP Molecular chaperones in the matrix exert multiple functions in translocation, sorting, folding, and assembly of newly imported proteins

Protein import into mitochondria.

Lipids of mitochondria

https://epub.ub.uni-muenchen.de/7513/1/Neupert_Walter_7513.pdf

Mitochondrial protein import.

MITOCHONDRIA take up and extrude various inorganic and organic ions, as well as larger substances such as proteins1–4. The technique of patch clamping should provide real-time information on such transport and on energy transduction in oxidative phosphorylation. It has been applied to detect microscopic currents from mitochondrial membranes and conductances of ion channels in the 5–1,000 pS range in the outer and inner membranes5–10. These pores are not, however, selective for particular ions. Here we use fused giant mitoplasts prepared from rat liver mitochondria to identify a small conductance channel highly selective for K+ in the inner mitochondrial membrane. This channel can be reversibly inactivated by ATP applied to the matrix side under inside-out patch configuration; it is also inhibited by 4-aminopyridine and by glybenclamide. The slope conductance of the unitary currents measured at negative membrane potentials was 9.7±1.0pS (mean ± s.d., n = 6) when the pipette solution contained 100 mM K+ and the bathing solution 33.3 mM K+. Our results indicate that mitochondria depolarize by generating a K+ conducta-ance when ATP in the matrix is deficient.

ATP-sensitive K+ channel in the mitochondrial inner membrane

How mitochondria import proteins

The precursor of cytochrome b(2) (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b(2) precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The yeast mitochondrial outer membrane was isolated and 10 of its major polypeptides were identified (mol. wts. 109, 70, 57, 45, 45, 42, 33, 29, 25 and 14 kd). The membrane has no major polypeptide in common with either mitochondrial inner membrane or rough microsomes. Protease treatment and immunochemical techniques showed that virtually all of the isolated outer membrane vesicles are sealed and display the same surface orientation as in the intact mitochondrion.

The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles.

Publisher Summary This chapter outlines a general approach for examining the import of a given mitochondrial protein and emphasizes some of the rationale and the methods available for doing so In examining the import of a particular mitochondrial protein, some questions must be answered as to whether the protein is initially synthesized as a larger precursor; the polypeptide can be taken up by mitochondria posttranslationally; import of the protein is mediated by binding to a mitochondrial outer membrane receptor; and import of the polypeptide require an energized inner membrane The import of polypeptides into the mitochondrial matrix, the inner membrane, or the intermembrane space requires an electrochemical gradient across the mitochondrial inner membrane If an imported mitochondrial polypeptide is made as a larger precursor, then the energy dependence of import is conveniently checked by demonstrating accumulation of this precursor in yeast cells pulse-labeled in the presence, but not in the absence, of 20μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) This assay is unsuitable for precursors that have the same molecular weight as the mature protein

Rick Hay

Papers

How mitochondria import proteins

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles.

Assessing import of proteins into mitochondria: an overview.