Animal cells secrete small vesicles, otherwise known as exosomes and microvesicles (EMVs). A short, N-terminal acylation tag can target a highly oligomeric cytoplasmic protein, TyA, into secreted vesicles (Fang, Y., Wu, N., Gan, X., Yan, W., Morell, J. C., and Gould, S. J. (2007) PLoS Biol. 5, 1267-1283). However, it is not clear whether this is true for other membrane anchors or other highly oligomeric, cytoplasmic proteins. We show here that a variety of plasma membrane anchors can target TyA-GFP to sites of vesicle budding and into EMVs, including: (i) a myristoylation tag; (ii) a phosphatidylinositol-(4,5)-bisphosphate (PIP 2 )-binding domain; (iii), a phosphatidylinositol-(3,4,5)-trisphosphate-binding domain; (iv) a prenylation/palmitoylation tag, and (v) a type-1 plasma membrane protein, CD43. However, the relative budding efficiency induced by these plasma membrane anchors varied over a 10-fold range, from 100% of control (AcylTyA-GFP) for the myristoylation tag and PIP 2 -binding domain, to one-third or less for the others, respectively. Targeting TyA-GFP to endosome membranes by fusion to a phosphatidylinositol 3-phosphate-binding domain induced only a slight budding of TyA-GFP, ϳ2% of control, and no budding was observed when TyA-GFP was targeted to Golgi membranes via a phosphatidylinositol 4-phosphate-binding domain. We also found that a plasma membrane anchor can target two other highly oligomeric, cytoplasmic proteins to EMVs. These observations support the hypothesis that plasma membrane anchors can target highly oligomeric, cytoplasmic proteins to EMVs. Our data also provide additional parallels between EMV biogenesis and retrovirus budding, as the anchors that induced the greatest budding of TyA-GFP are the same as those that mediate retrovirus budding.Animal cells release single membrane vesicles that have the same topology as the cell and a variable diameter of ϳ50 -250 nm (1-8). These vesicles mediate the secretion of a wide variety of proteins, lipids, mRNAs, and micro RNAs, interact with neighboring cells, and can thereby transmit signals, proteins, lipids, and nucleic acids from cell to cell (3, 9 -11). Furthermore, the ability of vesicle-derived nucleic acids to alter gene expression in neighboring cells makes it likely that secreted vesicles can fuse with neighboring cells in a nonviral pathway of intercellular vesicle traffic. Not surprisingly, there is increasing evidence that secreted vesicles play important roles in normal physiological processes (e.g. immune signaling (2), development (12, 13) and in human disease (e.g. cancer (14 -16), amyloidopathies (17-19), and viral infections (4, 7, 20 -22)).Current models posit that there might be two separate pathways for the formation of small secreted vesicles: microvesicle biogenesis, which involves vesicle budding directly from the plasma membrane (PM) 2 ; and exosome biogenesis, which involves vesicle budding into endosomes to form multivesicular bodies (MVBs), followed by MVB-PM fusion (3,8). However, differentiating between microvesic...

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