Identification of two alternate splice variants of a novel serine protease expressed in steroidogenic tissues.
During the search for the serine protease that cleaves pro-gamma-melatropin to stimulate adrenal growth, we identified another novel protease, which we called Adrenal mitochondrial protease (AmP). In situ hybridisation detected AmP transcripts in steroidogenic tissues such as the brain, testis, in ovarian follicles as well as in the adrenal cortex. Full length cloning identified two splice variants differing by a 222 nucleotide insertion in the 5' end of the short variant. The shorter variant codes for a 371 amino acid protein of 40.7 kDa and computer analysis predicts it to be targeted to the cytosol while the longer 445 amino acid protein of 48.4 kDa is mitochondrial. Cellular targeting was confirmed by tagging with GFP. The short variant was clearly cytosolic however, the cells expressing AmP-Long had large vacuoles, possibly as a result of distended (apoptotic?) mitochondria. Due to the mitochondrial localisation of the long variant of the protease and its expression in steroidogenic tissues, it may be expected to be involved in the steroidogenic pathway, possibly by cleaving steroidogenic acute regulatory protein (StAR). We investigated this by co-transfecting AmP-Long with StAR and F2 plasmid into COS-1 cells and measuring the effect on pregnenolone production. It was found that AmP-Long has no effect on steroidogenesis nor cleaves StAR as was shown by western blot analysis using StAR antibody.
Omer
Selma
S
School of Animal and Microbial Sciences, The University of Reading, Whiteknights, P.O. Box 228, Reading RG6 6AJ, UK.
Lomthaisong
Khomsorn
K
Bicknell
Andrew B
AB
D000311:Adrenal Glands / Q000201:enzymology; D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular; D014644:Genetic Variation; D008565:Membrane Proteins; D008928:Mitochondria / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000187:drug effects; D011284:Pregnenolone / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D014018:Tissue Distribution; D000098682:Steroidogenic Acute Regulatory Protein
D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D000098682:Steroidogenic Acute Regulatory Protein; D011284:Pregnenolone; D012697:Serine Endopeptidases; C450831:TMPRSS5 protein, human