Permeabilization of mammalian cells to proteins: poliovirus 2A(pro) as a probe to analyze entry of proteins into cells.
Two hybrid protein molecules containing the poliovirus protease 2A (MBP-2A(pro)) (maltose-binding protein-2A(pro) and MBP-Pseudomonas exotoxin A-2A(pro)) have been constructed and purified. Both hybrid proteins efficiently cleave the translation initiation factor eIF-4G when they are co-internalized into cells with adenovirus particles. Almost no intact eIF-4G can be detected in cells incubated with these proteins following this method. Reovirus infectious subviral particles also promote the delivery of MBP-2A(pro) into cells, although less efficiently than adenovirus particles. None of the other methods employed to permeabilize cells to MBP-2A(pro) achieves the degree of eIF-4G cleavage observed with adenovirus particles. By comparison about 30% of cells electroporated with MBP-2A(pro) still contain intact eIF-4G. More drastic electroporation conditions lead to a significant decrease of cell survival. Osmotic lysis of pinocytic vesicles resulted in 30% of the eIF-4G being cleaved in cells treated in suspension. Delivery of MBP-2A(pro) by pH-sensitive liposomes leads to poor hydrolysis of eIF-4G. Taken together our results indicate that permeabilization of cells with adenovirus particles is the most efficient method for introducing MBP-2A(pro) into cells.
Novoa
I
I
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma de Madrid, Canto Blanco, Spain.
Benavente
J
J
Cotten
M
M
Carrasco
L
L
D036002:ADP Ribose Transferases; D000256:Adenoviridae; D001427:Bacterial Toxins; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000493:pharmacokinetics*; D002463:Cell Membrane Permeability; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000493:pharmacokinetics*; D018274:Electroporation; D005098:Exotoxins / Q000235:genetics / Q000493:pharmacokinetics*; D006367:HeLa Cells; D006801:Humans; D058619:Maltose-Binding Proteins; D015335:Molecular Probes; D009997:Osmotic Pressure; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014764:Viral Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D002352:Carrier Proteins; D003546:Cysteine Endopeptidases; D005098:Exotoxins; D058619:Maltose-Binding Proteins; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C057400:picornain 2A, Picornavirus