Subtilisin kexin isozyme-1 (SKI-1)/site 1 protease is a mammalian subtilase composed of distinct functional domains. Among the major substrates of SKI-1 are the sterol regulatory element-binding proteins, regulating cholesterol and fatty acid homeostasis. Other substrates include the stress response factor activating transcription factor-6, the brain-derived neurotrophic factor, and the surface glycoproteins of highly infectious viruses belonging to the family of Arenaviridae. Domain deletion and/or point mutants were used to gauge the role of the various domains of SKI-1. Biosynthesis, cellular trafficking, and sterol regulatory element-binding protein-2 cleavage activity were used as diagnostic tools. Results revealed that Arg(130) and Arg(134) are critical for the autocatalytic primary processing of the prosegment and for the subsequent efficient exit of SKI-1 from the endoplasmic reticulum. Functional mapping of the growth factor cytokine receptor motif suggested a folding role within the endoplasmic reticulum. Microsequencing of the remaining membrane-bound stub following ectodomain shedding of SKI-1 localized the shedding site to KHQKLL(953) downward arrow. Site-directed mutagenesis, in vitro cleavage of a synthetic peptide containing the shedding site, and inhibitor studies favor an autocatalytic event occurring at a non-canonical SKI-1 recognition sequence, with P2 and P1 Leu being very critical. In conclusion, multiple domains ensuring optimal functional characteristics control SKI-1 activity and cellular trafficking.