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J. Biol. Chem., Vol. 282, Issue 37, 27402-27413, September 14, 2007

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The Proprotein Convertase SKI-1/S1P

ALTERNATE TRANSLATION AND SUBCELLULAR LOCALIZATION^*

Philomena Pullikotil, Suzanne Benjannet, Janice Mayne, and Nabil G. Seidah¹

From the Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada and Hormones, Growth, and Development, Ottawa Health Research Institute, The Ottawa Hospital, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada

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 KHQKLL⁹⁵³, 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 Met⁹⁹⁰ to M990A, M990I, and M990L failed to generate St-2, suggesting an internal alternate translation event at Met⁹⁹⁰, 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.

Received for publication, April 16, 2007 , and in revised form, July 2, 2007.

^* This work was supported by Canadian Institutes of Health Research Grant MOP-36496, by a Canada Chair, and by a private donation from the Strauss Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Ave. W., Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5609; Fax: 514-987-5542; E-mail: seidahn{at}ircm.qc.ca.

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