The proprotein convertase SKI-1/S1P: alternate translation and subcellular localization.
Subtilisin kexin isozyme-1 (SKI-1) represents the first mammalian member of secretory subtilisin-like processing enzymes that cleaves after nonbasic residues. It is synthesized as an inactive precursor that undergoes three sequential autocatalytic processing steps of its N-terminal prosegment and an ectodomain shedding at a site near the transmembrane domain. The various cellular functions of SKI-1 emphasize the need to understand the sites of its activation and shedding. We have previously shown that SKI-1 undergoes autocatalytic shedding at the sequence KHQKLL(953) downward arrow, resulting in a membrane-bound stump called St-1 (amino acids 954-1052). However, little is known about the cellular localization of SKI-1 or its shed forms. In the present study, we have further identified a smaller C-terminal fragment St-2 generated closer to the transmembrane domain. By sequencing and mass spectrometric analysis, the start site and the molecular mass of St-2 were determined. Site-directed mutagenesis revealed the critical amino acid involved in this novel process. Mutation of Met(990) to M990A, M990I, and M990L failed to generate St-2, suggesting an internal alternate translation event at Met(990), as confirmed by an in vitro transcription/translation assay. Confocal microscopy defined the subcellular localization of SKI-1 and its fragments. The data show that most of membrane-bound SKI-1 and its stumps St-1 and St-2 localize to the Golgi and can enter the endosomal/lysosomal compartments but do not sort to the cell surface. Deletion studies showed that the transmembrane domain of SKI-1 determines its trafficking. Finally, rSt-1 and rSt-2 seem to affect the processing of ATF6 by SKI-1, but cellular stress does not regulate the production of St-2.
Pullikotil
Philomena
P
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada and.
Benjannet
Suzanne
S
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada and.
Mayne
Janice
J
Hormones, Growth, and Development, Ottawa Health Research Institute, The Ottawa Hospital, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada.
Seidah
Nabil G
NG
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada and. Electronic address: seidahn@ircm.qc.ca.
D051702:Activating Transcription Factor 6 / Q000096:biosynthesis; D000595:Amino Acid Sequence; D002478:Cells, Cultured; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D043484:Proprotein Convertases / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis; D012697:Serine Endopeptidases / Q000032:analysis / Q000737:chemistry / Q000235:genetics*
C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C115575:membrane-bound transcription factor peptidase, site 1