Cellular internalization of green fluorescent protein fused with herpes simplex virus protein VP22 via a lipid raft-mediated endocytic pathway independent of caveolae and Rho family GTPases but dependent on dynamin and Arf6.
VP22 is a structural protein of the herpes simplex virus and has been reported to possess unusual trafficking properties. Here we examined the mechanism of cellular uptake of VP22 using a fusion protein between the C-terminal half of VP22 and green fluorescent protein (GFP). Adsorption of VP22-GFP onto a cell surface required heparan sulfate proteoglycans and basic amino acids, in particular, Arg-164 of VP22. Inhibitor treatment, RNA interference, expression of dominant-negative mutant genes, and confocal microscopy all indicated that VP22-GFP enters cells through an endocytic pathway independent of clathrin and caveolae but dependent on dynamin and Arf6 activity. As with CD59 (a lipid raft marker), cell-surface VP22-GFP signals were resistant to Triton X-100 treatment but only partially overlapped cell-surface CD59 signals. Furthermore, unlike other lipid raft-mediated endocytic pathways, no Rho family GTPase was required for VP22-GFP internalization. Internalized VP22 initially entered early endosomes and then moved to lysosomes and possibly recycling endosomes.
Nishi
Kenji
K
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: kennishi@biochem.s.u-tokyo.ac.jp.
Saigo
Kaoru
K
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: saigo@biochem.s.u-tokyo.ac.jp.
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D021941:Caveolae / Q000502:physiology*; D002738:Chloroquine / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D034281:Dynamins / Q000502:physiology*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D021962:Membrane Microdomains / Q000502:physiology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000502:physiology*
D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D049452:Green Fluorescent Proteins; D002738:Chloroquine; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020741:rho GTP-Binding Proteins; D034281:Dynamins