EXPRESSION OF MUTANT DYNAMIN INHIBITS TOXICITY AND TRANSPORT OF ENDOCYTOSED RICIN TO THE GOLGI-APPARATUS

Endocytosis and intracellular transport of ricin were studied in stabl e transfected HeLa cells where overexpression of wild-type (WT) or mut ant dynamin is regulated by tetracycline. Overexpression of the temper ature-sensitive mutant dyn(G273D) at the nonpermissive temperature or the dyn(K44A) mutant inhibits clathrin-dependent endocytosis (Damke, H ., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131:69-80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915-934). Under these conditions, ricin was endocytose d at a normal level, Surprisingly, overexpression of both mutants made the cells less sensitive to ricin, Butyric acid and trichostatin A tr eatment enhanced dynamin overexpression and increased the difference i n toxin sensitivity between cells with normal and mutant dynamin. Into xication with ricin seems to require toxin transport to the Golgi appa ratus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949-966), and this process was monitored by measuring the incorporation of radi oactive sulfate into a modified ricin molecule containing a tyrosine s ulfation site, The sulfation of ricin was much greater in cells expres sing dyn(WT) than in cells expressing dyn(K44A). Ultrastructural analy sis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dyn(K44A). In con trast, ricin transport to lysosomes as measured by degradation of I-12 5-ricin was essentially unchanged in cells expressing dyn(K44A). These data demonstrate that although ricin is internalized by clathrin-inde pendent endocytosis in cells expressing mutant dynamin, there is a str ong and apparently selective inhibition of ricin transport to the Golg i apparatus. Also, in cells with mutant dynamin, there is a redistribu tion of the mannose-6-phosphate receptor.