Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin͞kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin͞kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT2SL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca 2؉ -dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brainderived neurotrophic factor.

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Much is known about ␤ 2 -adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the ␤ 2 -adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-G␤␥ complexes initially associate in the endoplasmic reticulum, whereas G␣ subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.