monensin and tetrafluoroaluminate

An active absorption of polypeptides (elcatonin = CCT; adrenocorticotropic hormone) had been previously observed in the nasal respiratory mucosa of the rabbit. Its saturation kinetics and the parallel absence of a net transfer of other non-polypeptidic organic markers excluded the involvement of a simple pinocytosis. This absorption has been now better localized and further characterized. Unidirectional CCT fluxes (determined with radioimmunoassay) have been concomitantly monitored with transepithelial electric potential difference (Vms). Although the mucosae covering the ectoturbinal A and the lower and upper conchae displayed similar Vms, the active CCT transport was only evidenced in the upper concha. In this region cytochalasin B (which by disassembling actin microfilaments prevents the apical formation of vesicles in epithelial cells) and monensin (which prevents the split of the ligand-receptor complex in the endosomes) both eliminated the net CCT absorption, however, also permanently increasing the passive CCT junctional permeability. Aluminum fluoride (which prevents the fusion of endocytic vesicles into endosomes) and colchicine (which disrupts microtubules along which vesicles move in the cytoplasm) also permanently abolished net CCT transport, without affecting, or shortly and transiently affecting, passive permeability. On the whole these results are in favor of an active CCT transport supported by a specific vesicular transport.

Topics: Aluminum Compounds; Animals; Biological Transport, Active; Calcitonin; Colchicine; Cytochalasin B; Endocytosis; Fluorides; In Vitro Techniques; Intestinal Absorption; Kinetics; Male; Monensin; Nasal Mucosa; Peptides; Pinocytosis; Rabbits

Recent studies suggest that a cycle of acylation/deacylation is involved in the vesicular transport of proteins between intracellular compartments at both the budding and the fusion stage (Glick, B. S., and J. E. Rothman. 1987. Nature (Lond.). 326:309-312). Since a number of cellular processes requiring vesicular transport are inhibited during mitosis, we examined the fatty acylation of proteins in interphase and mitotic cells. We have identified a major palmitoylated protein with an apparent molecular weight of 62,000 (p62), whose level of acylation increases 5-10-fold during mitosis. Acylation was reversible and p62 was no longer palmitoylated in cells that have exited mitosis and entered G1. p62 is tightly bound to the cytoplasmic side of membranes, since it was sensitive to digestion with proteases in the absence of detergent and was not removed by treatment with 1 M KCl. p62 is removed from membranes by nonionic detergents or concentrations of urea greater than 4 M. The localization of p62 by subcellular fractionation is consistent with it being in the cis-Golgi or the cis-Golgi network. A palmitoylated protein of the same molecular weight was also observed in interphase cells treated with inhibitors of intracellular transport, such as brefeldin A, monensin, carbonylcyanide m-chlorophenylhydrazone, or aluminum fluoride. The protein palmitoylated in the presence of brefeldin A was shown to be the same as that palmitoylated during mitosis using partial proteolysis. Digestion with two enzymes, alkaline protease and endoprotease lys-C, generated the same 3H-palmitate-labeled peptide fragments from p62 from mitotic or brefeldin A-treated cells. We suggest that the acylation and deacylation of p62 may be important in vesicular transport and that this process may be regulated during mitosis.

Topics: Acylation; Aluminum; Aluminum Compounds; Animals; Anti-Bacterial Agents; Brefeldin A; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Fractionation; Centrifugation, Density Gradient; CHO Cells; Clone Cells; Cricetinae; Cyclopentanes; Fluorides; Fluorine; Interphase; Microsomes; Mitosis; Mitotic Index; Molecular Weight; Monensin; Nocodazole; Organelles; Palmitic Acid; Palmitic Acids; Proteins; Subcellular Fractions