A stable HeLa cell line expressing a dynamin mutant, dyn ts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dyn ts carries a point mutation, G273D, corresponding to the Drosophila shibire tsl allele. The ts-defect in receptormediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wildtype levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin-and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dyn ts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway.

YNAMIN is a 100-kD GTPase originally isolated as a nucleotide-dependent microtubule binding protein (Shpetner and Vallee, 1989). Dynamin function in vivo has been studied using transiently (Herskovits et al., 1993; van der Bliek et al., 1993) and stably (Damke et al., 1994) transfected cells overexpressing dominant interfering, GTPase-defective mutants. Receptor-mediated endocytosis is potently and specifically inhibited in stably transformed cells with inducible expression of a dynamin mutant (designated K44A and previously referred to as elel) defective in GTP binding (Damke et al., 1994). Phenotypic characterization of these cells established that dynamin function is required for the formation of constricted coated pits at the plasma membrane. Importantly, no other vesicular trafficking events, including protein transport through the exocytic pathway, receptor recycling through the endosomal pathway or clathrin-coated vesicle-mediated sorting of lysosomal hydrolases were affected (Damke et al., 1994). Consistent with this specificity of function and even when overexpressed> 20-fold, membrane-associated dynamin is exclusively localized to clathtin-coated pits on the plasma membrane (Damke et al.,