Inhibition of Secretion by 1,3-Cyclohexanebis(methylamine), a Dibasic Compound That Interferes with Coatomer Function

Abstract

We noted previously that certain aminoglycoside antibiotics inhibit the binding of coatomer to Golgi membranes in vitro. The inhibition is mediated in part by two primary amino groups present at the 1 and 3 positions of the 2-deoxystreptamine moiety of the antibiotics. These two amines appear to mimic the epsilon-amino groups present in the two lysine residues of the KKXX motif that is known to bind coatomer. Here we report the effects of 1, 3-cyclohexanebis(methylamine) (CBM) on secretion in vivo, a compound chosen for study because it contains primary amino groups that resemble those in 2-deoxystreptamine and it should penetrate lipid bilayers more readily than antibiotics. CBM inhibited coatomer binding to Golgi membranes in vitro and in vivo and inhibited secretion by intact cells. Despite depressed binding of coatomer in vivo, the Golgi complex retained its characteristic perinuclear location in the presence of CBM and did not fuse with the endoplasmic reticulum (ER). Transport from the ER to the Golgi was also not blocked by CBM. These data suggest that a full complement of coat protein I (COPI) on membranes is not critical for maintenance of Golgi integrity or for traffic from the ER to the Golgi but is necessary for transport through the Golgi to the plasma membrane.

A role for the host coatomer and KDEL receptor in early vaccinia biogenesis

“…A major set of these interactions could be feasibly screened by an in vivo approach using a pharmacologic agent, 1,3-cyclohexanebis-[methylamine] (CBM), which disrupted the interaction of coatomer with proteins that contained di-basic residues (12). When infected cells were treated with CBM, vaccinia replication was reduced by Ϸ15-fold (Fig.…”

“…Notably, we used a dose of CBM (2 mM) that was shown to be relatively specific in disrupting the binding of coatomer to di-basic residues (12). However, because such a dose of CBM was predicted not to disrupt the binding of coatomer with its targets completely (12), this circumstance further high- (E) HeLa cells, either treated with siRNA against ␤-COP for 48 h or mock-treated, were then infected with WR virus for 24 h. Intracellular viral forms were then purified from infected cells, followed by EM examination for intact viruses. Quantitation was performed from 10 randomly selected images, and then expressed as mean with standard error (P Ͻ 0.01).…”

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

“…S4B). We then used 1,3-cyclohexanebismethylamine (CBM), an inhibitor of COPI function (32,33), to examine involvement of COPI in VSV gene expression. Treatment of cells with CBM 1 h before infection reduced VSV M protein levels in a dosedependent manner (Fig.…”

Evidence that the transport of ricin to the cytoplasm is independent of both Rab6A and COPI

“…The effect of Rab6A-T27N on the transport of influenza virus hemagglutinin protein (HA) from the ER to the cell surface was measured by incorporation of radioactivity from Tran 35 S-label into HA as previously described (Hu et al, 1999). In brief, cells were transfected with plasmid encoding wild-type Rab6A or the mutant Rab6A-T27N one day before an experiment.…”

“…Protein synthesis was measured by the incorporation of radioactivity from Tran 35 S-label into acid insoluble protein essentially as described previously (Hu et al, 1999). Cells were transfected as indicated in Table legends and a toxin was added at different concentrations for the indicated times.…”