Cell surface adherence and endocytosis of protein transduction domains.
Protein transduction domains (PTD), such as the HIV TAT and the herpes simplex virus VP22 proteins, are reported to translocate across the membranes of mammalian cells. The mechanism of PTD membrane translocation has largely remained elusive, but recent studies suggest that the reported PTD translocation is due to a fixation artifact. We have constructed and expressed the PTDs VP22, TAT, polyarginine, and polylysine fused to the green fluorescent protein to visualize these proteins in both living and fixed cells. The investigated PTDs strongly adhered to the surface of living cells and were internalized by constitutive endocytosis. No cytosolic or nuclear import of the proteins was detected. In contrast, the PTD-GFP fusion proteins were redistributed to the cytosol and nucleus directly after fixation. Our findings suggest that the PTDs only mediate cell surface adherence, a property shared with many other positively charged macromolecules. The cell surface adherence results in endocytosis and accumulation of proteins in endosomes. We suggest that the biological effects observed for PTD fusion proteins are due to cell surface interactions and internalization of the proteins into cells by classical endocytosis.
Lundberg
Mathias
M
Division of Clinical Virology F68, Karolinska Institute, Huddinge University Hospital, S-14186, Stockholm, Sweden.
Wikström
Sara
S
Johansson
Magnus
M
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015678:Viral Structural Proteins / Q000378:metabolism
D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D049452:Green Fluorescent Proteins; C015462:polyarginine