Protein delivery with transportans is mediated by caveolae rather than flotillin-dependent pathways.
Delivery of large bioactive cargoes into cells with the help of cell-penetrating peptides (CPPs) is mostly based on endocytic processes. Here we map the cellular pathways used by transportan and transportan 10 (TP10) for protein transduction in HeLa cells. CPP-mediated cellular delivery is often suggested to be lipid-raft-dependent; therefore, we used flotillin-1, caveolin, Rab5, and PI3P as markers to elucidate the involvement of these particular endosomal pathways in the protein uptake process. Confocal laser scanning and electron microscopy reveal only a negligible overlap of avidin/neutravidin conveyed into cells by transportans with the raft marker flotillin-1 or early endosomal markers Rab5 and PI3P. However, about 20% of protein-CPP complexes colocalize with the caveolar/caveosomal marker caveolin, and down-regulation of caveolin-1 by siRNA treatment leads to the inhibition of the CPP-mediated protein uptake by 30-50%. On the contrary, the lack of flotillin-1 increases rather than decreases the CPP-mediated protein transport. The participation of the caveolin-1-dependent pathway in CPP-mediated protein delivery was also corroborated by using caveolin-1 knockout mouse embryonic fibroblasts.
Säälik
Pille
P
Institute of Molecular and Cell Biology, University of Tartu, Estonia.
Padari
Kärt
K
Niinep
Aira
A
Lorents
Annely
A
Hansen
Mats
M
Jokitalo
Eija
E
Langel
Ulo
U
Pooga
Margus
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000172:deficiency / Q000235:genetics; D002784:Cholesterol / Q000378:metabolism; D015536:Down-Regulation; D011992:Endosomes / Q000378:metabolism; D019004:Galanin / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D014862:Wasp Venoms / Q000737:chemistry / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D051242:Caveolin 1; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014862:Wasp Venoms; C106354:flotillins; C055525:phosphatidylinositol 3-phosphate; C110068:transportan; D019004:Galanin; D002784:Cholesterol; D020696:rab5 GTP-Binding Proteins