Preferential processing of the S1 subunit of pertussis toxin that is bound to eukaryotic cells.
Labelled [125I]-pertussis toxin was prepared and used to measure the association of pertussis toxin (PT) to eukaryotic cells. PT was radioiodinated by the lactoperoxidase method which preferentially radioiodinated the S1 subunit. PT was radioiodinated at a high specific activity and possessed the same cytotoxicity as native PT as demonstrated by the ability to cluster Chinese hamster ovary (CHO) cells. Cell association of [125I]-PT was not inhibited by excess non-radiolabelled PT, which indicated that the initial interaction between PT and CHO cells involved a large number of low-affinity receptors. At 37 degrees C, the S1 within cell-associated PT was preferentially processed to an S1 with a lower apparent molecular weight (termed S1p). This processing was inhibited by the addition of unlabelled PT, indicating that the processing event was saturable and specific. S1 processing occurred in CHO, Madin-Darby canine kidney (MDCK) cells, and pig kidney (LLC-PK1) cells. A pulse-chase experiment showed that, at 37 degrees C but not at 22 degrees C, essentially all of the cell-associated S1 was processed within 3 h of a chase. Reagents that were previously shown to inhibit the ability of PT to ADP-ribosylate Gi proteins in intact CHO cells also inhibited the preferential processing of S1 within cell-associated PT, in the order of efficiency: 22 degrees C > chloroquine > nocodazole > brefeldin A. This indicates that S1 processing requires an early endosomal function.
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
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Finck-Barbançon
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JT
Barbieri
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D016466:CHO Cells; D002451:Cell Compartmentation; D002738:Chloroquine / Q000494:pharmacology; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D004285:Dogs; D011992:Endosomes / Q000378:metabolism; D005057:Eukaryotic Cells; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008954:Models, Biological; D015739:Nocodazole / Q000494:pharmacology; D037342:Pertussis Toxin; D011402:Pronase / Q000494:pharmacology; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism; D013552:Swine; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D003517:Cyclopentanes; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D010566:Virulence Factors, Bordetella; C093426:pertussis toxin receptor; D020126:Brefeldin A; D002738:Chloroquine; D037342:Pertussis Toxin; D011402:Pronase; D015739:Nocodazole