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Expression of the Longin domain of TI‐VAMP impairs lysosomal secretion and epithelial cell migration
Author(s) -
ProuxGillardeaux Véronique,
Raposo Graça,
Irinopoulou Theano,
Galli Thierry
Publication year - 2007
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1042/bc20060097
Subject(s) - biology , secretion , microbiology and biotechnology , domain (mathematical analysis) , expression (computer science) , cell , computational biology , biochemistry , computer science , mathematical analysis , mathematics , programming language
Background information . TI‐VAMP (tetanus neurotoxin‐insensitive vesicle‐associated membrane protein; also called VAMP7) belongs to the Longin subfamily of v‐SNAREs (vesicular soluble N ‐ethylmaleimide‐sensitive fusion protein‐attachment protein receptors). The regulatory N‐terminal extension, called the Longin domain, of TI‐VAMP has been shown previously to have a dual biochemical function: it inhibits the capacity of TI‐VAMP to form SNARE complexes and it binds to the δ subunit of the AP‐3 (adaptor protein 3) complex in early endosomes, thereby targeting TI‐VAMP to late endosomes. Results . We have generated MDCK (Madin—Darby canine kidney) cell lines expressing the Longin domain of TI‐VAMP coupled to GFP (green fluorescent protein) in a doxycycline‐dependent manner. As expected, AP‐3δ (AP‐3 δ subunit) is not properly localized in Longin‐expressing cells. We have shown that the expression of the Longin domain impairs lysosomal secretion, as determined by the release of a pre‐internalized fluorescent fluid‐phase marker and by electron microscopy of the membrane‐associated released particles. Membrane repair following mechanical wounding, a process requiring lysosomal secretion, is also impaired in cells expressing the Longin domain. Furthermore, cell migration, assessed by wound healing of MDCK monolayers, is also inhibited. Conclusions . The results of the present study suggest that the expression of the Longin domain of TI‐VAMP regulates lysosomal secretion of epithelial cells and provide molecular evidence for a role of the late endocytic system in cell migration.