title: Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein.
abstract: Recent studies of vascular adhesion protein-1 (VAP-1) have greatly advanced our understanding of the important role this protein plays in the establishment and progression of inflammatory disease. To facilitate more detailed studies on the function of VAP-1, we developed a GFP-fusion protein that enabled us to monitor the trafficking of the protein in three selected cell types: hepatic sinusoidal endothelial cells, liver myofibroblasts and an hepatic stellate cell line (LX-2). The fusion protein was detected as punctate cytoplasmic GFP staining, but was present only at low levels at the cell surface in all cell types studied. The subcellular distribution of the protein was not altered in a catalytically inactive mutant form of the protein (Tyr471Phe) or in the presence of exogenous VAP-1 substrate (methylamine) or inhibitor (semicarbazide). The GFP-VAP-1 protein was localized to the Golgi apparatus (GM-130), endoplasmic reticulum (GRP94) and early endosomes (EEA-1). Additional staining for VAP-1 revealed that the overexpressed protein was also present in vesicles that were negative for GFP fluorescent signal and did not express EEA-1. We propose that these vesicles are responsible for recycling the fusion protein and that the fluorescence of the GFP moiety is quenched at the low pH within these vesicles. This feature of the protein makes it well suited for live cell imaging studies where we wish to track protein that is being actively trafficked within the cell in preference to that which is being recycled.
authors.affiliation: Centre for Liver Research and NIHR Biomedical Research Unit, Institute of Biomedical Research, MRC Centre for Immune Regulation, College of Medicine and Dentistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. c.j.weston@bham.ac.uk
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Weston
authors.forename: Emma L
authors.initials: EL
authors.lastname: Shepherd
authors.forename: David H
authors.initials: DH
authors.lastname: Adams
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000235:genetics / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005021:Ethylamines / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000166:cytology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D006631:Amine Oxidase (Copper-Containing); D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D005021:Ethylamines; D049452:Green Fluorescent Proteins; D051907:Lysosomal Membrane Proteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C093846:early endosome antigen 1; C004504:2-bromoethylamine; C076668:AOC3 protein, human