The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4.
The mechanisms governing the fast, regulated exocytosis of enlargeosomes have been unknown, except for the participation of annexin-2 in a pre-fusion step. We investigated whether any SNAREs are involved. In PC12-27 cells, which are enlargeosome-rich, the expressed SNAREs exhibited various distributions (trans-Golgi network, scattered puncta, plasma membrane); however, only VAMP4 was colocalized in discrete puncta with the enlargeosome marker desmoyokin. The exocytosis of the organelle, revealed by capacitance increases and by surface appearance of desmoyokin, was largely inhibited by microinjection of anti-VAMP4, anti-syntaxin-6 and anti-SNAP23 antibodies, by incubation with botulinum toxin E, and by transfection of VAMP4 and syntaxin-6 siRNAs. Microinjection of the antibodies anti-VAMP7, anti-VAMP8 and anti-syntaxin-4, and transfection with the VAMP8 siRNA were ineffective. Inhibition of enlargeosome exocytosis by VAMP4 siRNA also occurred in a cell type that was competent for neurosecretion, SH-SY5Y. Moreover, in cells expressing a VAMP4-GFP construct, enlargeosome exocytosis and surface appearance of fluorescence occurred concomitantly, and many ensuing surface patches were co-labelled by GFP and desmoyokin. VAMP4, an R-SNARE that has never been shown to participate in regulated exocytoses, therefore appears to be harboured in the membrane of enlargeosomes and to be a member of the machinery mediating their regulated exocytosis. Syntaxin-6 and SNAP23 appear also to be needed for the process to occur; however, the mechanism of their participation, whether direct or indirect, remains undefined.
Cocucci
Emanuele
E
Vita-Salute San Raffaele University, Center of Excellence in Cell Development, via Olgettina 58, 20132 Milan, Italy.
Racchetti
Gabriella
G
Rupnik
Marjan
M
Meldolesi
Jacopo
J
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism*
C489573:Ahnak protein, rat; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C533192:VAMP4 protein, rat