Mitochondrial porin can be translocated across both endoplasmic reticulum and mitochondrial membranes.
Mitochondrial porin is a major integral membrane protein of the outer membrane. To assess the stop-transfer sequence in the yeast porin molecule (P), we constructed the following chimeric proteins. (i) The signal sequence of interleukin 2, a secretory protein, was fused to the amino-terminus of porin (SP). (ii) The matrix targeting presequence of cytochrome c oxidase subunit IV was fused to the amino-terminus of porin (CP). (iii) The amino-terminal segment consisting of 42 amino acid residues of "70 kDa protein" of yeast mitochondria, a major membrane protein of the outer membrane, was introduced into the middle portion of interleukin 2 (IL70). These chimeric proteins were expressed with an in vitro transcription-translation system and their integration into microsomal membrane or mitochondrial membranes was examined. When the proteins were synthesized in vitro with wheat germ cell-free system in the presence of rough microsomal membrane (RM), SP was completely translocated across the membrane, processed by the signal peptidase, and glycosylated. The translocation of IL70 molecule across RM was stopped at the introduced amino-terminal segment of 70 kDa protein. The authentic porin did not interact with the microsomal membrane. To assess the interaction with mitochondria, porin and CP were synthesized with the reticulocyte lysate system and subjected to posttranslational import reaction with isolated rat liver mitochondria. The authentic porin was integrated into the outer membrane in an alkali-resistant fashion. CP was imported into the mitochondria and its presequence was cleaved by the processing protease in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)
Sakaguchi
M
M
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Hachiya
N
N
Mihara
K
K
Omura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002458:Cell Fractionation; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D007425:Intracellular Membranes / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D018272:Porins; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D018272:Porins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins