Mitochondrial import of Omi: the definitive role of the putative transmembrane region and multiple processing sites in the amino-terminal segment.
The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.
Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan.
Tsuyoshi
T
Kadomatsu
Masataka
M
Mori
Kazutoyo
K
Terada
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D051336:Mitochondrial Membranes / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection
D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2