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First published online 19 August 2008
doi: 10.1242/jcs.032029

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The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4

Emanuele Cocucci^1,*, Gabriella Racchetti², Marjan Rupnik³ and Jacopo Meldolesi^1,2,4,5,

¹ Vita-Salute San Raffaele University, Center of Excellence in Cell Development, via Olgettina 58, 20132 Milan, Italy
² Scientific Institute San Raffaele, via Olgettina 60, 20132 Milan, Italy
³ Institute of Physiology, Faculty of Medicine, University of Maribor, Slomskov, trg 15, 2000 Maribor, Slovenia
⁴ National Institute of Neurosciences, Italy
⁵ IIT Network, Research Unit of Molecular Neuroscience, via Olgettina 58, 20132 Milan, Italy

Author for correspondence (e-mail: meldolesi.jacopo{at}hsr.it)

Accepted 11 June 2008

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.

Key words: PC12 cells, SNAP23, Syntaxin-6, Ca²⁺, Ahnak

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