Abstract
Exosomes, 40–150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. Although exosomes are known to originate from maturation of endosomes into multivesicular endosomes (also known as multivesicular bodies) with subsequent fusion of the multivesicular endosomes with the plasma membrane, it remains unclear how cargos are selected for exosomal release. Using an inducible expression system for the exosome cargo protein GPRC5B and following its trafficking trajectory, we show here that newly synthesized GPRC5B protein accumulates in the Golgi complex prior to its release into exosomes. The L-type lectin LMAN2 (also known as VIP36) appears to be specifically required for the accumulation of GPRC5B in the Golgi complex and restriction of GPRC5B transport along the exosomal pathway. This may occur due to interference with the adaptor protein GGA1-mediated trans Golgi network-to-endosome transport of GPRC5B. The adaptor protein CD2AP-mediated internalization following cell surface delivery appears to contribute to the Golgi accumulation of GPRC5B, possibly in parallel with biosynthetic/secretory trafficking from the endoplasmic reticulum. Our data thus reveal a Golgi-traversing pathway for exosomal release of the cargo protein GPRC5B in which CD2AP facilitates the entry and LMAN2 impedes the exit of the flux, respectively.
Highlights
Exosomes, 40 –150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications
It is known that exosome biogenesis begins with the formation of multivesicular endosomes (MVEs; known as multivesicular bodies) [1, 2], which are characterized by harboring intraluminal vesicles (ILVs) that are derived from budding of the limiting endosomal membrane into the lumen of endosomes [21]
Using these methods of inducible expression and exosome preparation, we first noted that exosome release of GPRC5B-HA is tightly regulated by doxycycline in our cultured cell system with GPRC5B-HA being barely detectable in exosome fractions in the absence of doxycycline and the level increasing in response to doxycycline over time, first becoming detectable at 60 min after doxycycline addition and gradually accumulating thereafter (Fig. 1C)
Summary
40 –150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. A substantial fraction of GPRC5B-HA protein is still detectable in exosomes even when the CD2AP gene is completely inactivated (Fig. 3, C–E), suggesting that another mechanism, possibly involving the biosynthetic/secretory pathway, may work in parallel with CD2AP-dependent internalization of cell surface-localized GPRC5B-HA protein for exosomal release.
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