Protein sorting between mitochondrial outer and inner membranes. Insertion of an outer membrane protein into the inner membrane.
The amino terminal 29 amino acids of the outer mitochondrial membrane protein of yeast, OMM70 (MAS70), consisting of the targeting and membrane anchor domains, has been fused to a reporter protein, dihydrofolate reductase. The hybrid protein, designated pOMD29, was efficiently imported into the outer membrane of rat heart mitochondria by a process dependent on ATP and proteinase-sensitive components on the surface of the organelle, and in which the orientation of the native protein was retained. To determine if the protein translocation machinery of the inner membrane is also capable of recognizing and inserting pOMD29, direct access to the intermembrane space was provided to pOMD29 by selectively rupturing the mitochondrial outer membrane by osmotic shock. In this system, the outer membrane binding site for matrix-destined precursor proteins can be bypassed, and efficient import restored to proteinase-pretreated mitochondria. pOMD29 was imported into the inner membrane of osmotically-shocked mitochondria, mediated by protein components. The outer membrane orientation of pOMD29 was conserved when inserted into the inner membrane but, unlike the outer membrane, import into the inner membrane required delta psi. We conclude that the protein translocation machinery of the mitochondrial inner membrane is capable of recognizing and inserting a protein whose topogenic information otherwise results in insertion of the protein to the outer membrane. The significance of these findings for sorting of proteins between the mitochondrial inner and outer membranes is discussed.
Li
J M
JM
Department of Biochemistry, McGill University, Montreal, Canada.
Shore
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013367:Submitochondrial Particles / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C489039:TOM70 protein, S cerevisiae; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D013762:Tetrahydrofolate Dehydrogenase