Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors.
Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATPand temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.
Richard
Jean Philippe
JP
UMR 5124 CNRS, Université Montpellier 2, place Eugène Bataillon, 34095 Montpellier cedex 5, France.
Melikov
Kamran
K
Brooks
Hilary
H
Prevot
Paul
P
Lebleu
Bernard
B
Chernomordik
Leonid V
LV
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry*; D051242:Caveolin 1; D022461:Caveolins / Q000737:chemistry; D002460:Cell Line; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000378:metabolism; D005434:Flow Cytometry; D015696:Gene Products, tat / Q000737:chemistry*; D015756:Genes, tat; D006367:HeLa Cells; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008055:Lipids / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism; D008985:Monensin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013696:Temperature; D013997:Time Factors
D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; D015696:Gene Products, tat; D006497:Heparan Sulfate; D008055:Lipids; D008985:Monensin; D010455:Peptides; D011188:Potassium; C495540:CAV1 protein, human