Protein Targeting to Exosomes/Microvesicles by Plasma Membrane Anchors

Beiyi Shen,Ning Wu,Jr-Ming Yang,Stephen J Gould

doi:10.1074/jbc.m110.208660

Beiyi Shen, Ning Wu + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.208660

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Abstract

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, ∼2% 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.

Highlights

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]
PM Anchors and exosomes and microvesicles (EMVs) Targeting order oligomerization target proteins to HIV particles and are the primary budding information in the HIV Gag protein [7]. These data support the idea that retrovirus budding is a form of EMV biogenesis [4]. This relationship is reflected in the sites of EMV and retrovirus budding in different cell types; (i) macrophages bud HIV into MVB-like invaginations of the PM [25, 34], which closely resemble the structures that have been implicated in exosome biogenesis [2, 9, 34]; and (ii) T-cells bud both EMVs and HIV particles from discrete regions of the plasma membrane that are enriched for endosomal and exosomal markers and retain the endosomal ability to generate outward budding vesicles [23]
Our results confirm and extend the hypothesis that PM anchors can induce the budding of highly oligomeric cytoplasmic proteins, raise the possibility that endosomal membrane anchors cannot induce EMV cargo budding, and lend new support to the hypothesis that retrovirus budding is a form of EMV biogenesis

Summary

Introduction

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]. We reported previously that adding a 10-amino acid-long acyl tag, MGCINSKRKD-, to the N terminus of TyA-GFP results in its targeting to the PM, enrichment at endosome-like domains of the plasma membrane, and its secretion from the cell in EMVs [7].

Results

Conclusion