Farnesylation of CaaX-tagged diphtheria toxin A-fragment as a measure of transfer to the cytosol.
Diphtheria toxin binds to receptor-positive cells through its B-fragment, the toxin is then endocytosed, and the low pH in endosomes triggers the translocation of the enzymatically active A-fragment to the cytosol. A synchronous release of A-fragments into the cytosol can be induced by exposing cells with surface-bound toxin to low pH. We have used this protein translocation system to develop a novel method to study whether or not a protein is exposed to the cytosol. Protein farnesylation is a cytosolic modification signaled by a C-terminal CaaX motif, and to visualize the translocation process, we added a farnesylation signal to the toxin A-fragment. The A-fragment with an added CaaX motif was farnesylated within 1 h after exposure of cells with surface-bound toxin to low pH, and also A-fragment translocated from endosomes was quantitatively farnesylated. The results indicate that all cell-mediated reduction of the toxin implicates translocation of the A-fragment to the cytosol. The farnesylation was inhibited by lovastatin, the alkylating agent NEM, and the peptidomimetic farnesylation inhibitor B581. Farnesylated A-fragment partitioned preferentially into the detergent phase upon extraction with Triton X-114. Our data suggest that farnesylation of a CaaX tag is generally applicable as a cytosolic marker, and this strategy for monitoring protein transfer to the cytosol may have considerable potential for studying the transport to the cytosol of proteins added externally to cells.
Falnes
P O
PO
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Wiedłocha
A
A
Rapak
A
A
Olsnes
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003353:Corynebacterium diphtheriae / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D017830:Octoxynol; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols; D017368:Protein Prenylation; D014709:Vero Cells
D004167:Diphtheria Toxin; D010446:Peptide Fragments; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40