Rapid cellular removal of a membrane-inserted foreign polypeptide.
We have developed a system that makes it possible to study the fate of a foreign polypeptide that is inserted in the plasma membrane. Diphtheria toxin is a bacterial protein toxin that, upon acidification, has the ability to insert into the plasma membrane from the outside of eukaryotic cells. We present results that indicate endocytic uptake and degradation of the diphtheria toxin B-fragment after insertion into the membrane of Vero cells. The degradation rate of the fragment was found to be very high (t1/2 = 6 min) and dependent on cleavage of the extracellular part of the polypeptide with protease. Degradation was strongly inhibited in ATP-depleted cells, as well as at temperatures below 18 degrees C, and it was partially inhibited when the cytosol was acidified to block endocytosis from clathrin-coated pits. Degradation was also reduced in the presence of NH4Cl. The results indicate that the inserted and cleaved B-fragment is degraded by a process requiring endocytosis and transport to late endosomes or to lysosomes.
Moskaug
J O
JO
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Prydz
K
K
Olsnes
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003080:Cold Temperature; D004167:Diphtheria Toxin / Q000378:metabolism*; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D014709:Vero Cells
D004167:Diphtheria Toxin; D010446:Peptide Fragments; C039171:diphtheria toxin fragment B; D000643:Ammonium Chloride; D000255:Adenosine Triphosphate