VAP-33 localizes to both an intracellular vesicle population and with occludin at the tight junction.
Tight junctions create a regulated intercellular seal between epithelial and endothelial cells and also establish polarity between plasma membrane domains within the cell. Tight junctions have also been implicated in many other cellular functions, including cell signaling and growth regulation, but they have yet to be directly implicated in vesicle movement. Occludin is a transmembrane protein located at tight junctions and is known to interact with other tight junction proteins, including ZO-1. To investigate occludin's role in other cellular functions we performed a yeast two-hybrid screen using the cytoplasmic C terminus of occludin and a human liver cDNA library. From this screen we identified VAP-33 which was initially cloned from Aplysia by its ability to interact with VAMP/synaptobrevin and thus was implicated in vesicle docking/fusion. Extraction characteristics indicated that VAP-33 was an integral membrane protein. Antibodies to the human VAP-33 co-localized with occludin at the tight junction in many tissues and tissue culture cell lines. Subcellular fractionation of liver demonstrated that 83% of VAP-33 co-isolated with occludin and DPPIV in a plasma membrane fraction and 14% fractionated in a vesicular pool. Thus, both immunofluorescence and fractionation data suggest that VAP-33 is present in two distinct pools in the cells. In further support of this conclusion, a GFP-VAP-33 chimera also distributed to two sites within MDCK cells and interestingly shifted occludin's localization basally. Since VAP-33 has previously been implicated in vesicle docking/fusion, our results suggest that tight junctions may participate in vesicle targeting at the plasma membrane or alternatively VAP-33 may regulate the localization of occludin.
Lapierre
L A
LA
Departments of Internal Medicine and Cell Biology and the Yale Liver Center, Yale University, New Haven, CT, USA. lynnel@immag.mcg.edu.
Tuma
P L
PL
Navarre
J
J
Goldenring
J R
JR
Anderson
J M
JM
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism* / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D062793:Occludin; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D019108:Tight Junctions / Q000737:chemistry* / Q000502:physiology; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568987:Ocln protein, rat; C114051:VAPA protein, human; C491919:Vapa protein, rat; D033921:Vesicular Transport Proteins