Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

Transiently transfected CV-1 cells expressing Tac (no signal), the ID5 chimera (containing YQRL), or the R335D mutant (containing YQDL) were incubated for 30 min at 4~ with ]Zq-labeled anti-Tac mAb (Uchiyama et al., 1981) . Upon warming the cells to 370C, Tac was found to be poorly internalized (Fig. 7) , in agreement with the observations of Weissman et al. (1986) . The ID5 chimera was internalized at a rate of 3.5 % per minute (Fig. 7) , significantly faster than normal Tac, but slower than other internalized receptors with tyrosine-based motifs (Weissman et al., 1986; Hunziker et al., 1991; Casanova et al., 1991) . Interestingly, the R335D F/gum 9. Dimming of endogenous TGN38 staining by overexpressed chimeric constructs. (a and b) MOP-8 cells overexpressing T-G-G were stained with a mouse antibody to Tac and a rabbit antibody to endogenous TGN38, followed by a fluorescein-conjugated donkey antibody to mouse IgG and a rhodamine-conjugated donkey antibody to rabbit IgG. (e and f) MOP-8 cells overexpressing T-G-G were stained with a mouse antibody to Tac and a rabbit antibody to endogenous mannosidase II, followed by a fluorescein-conjugated donkey antibody to mouse IgG and a rhodami~-conjugated donkey antibody to rabbit IgG. (e) T-G-G, fluorescein channel; (f) mannosidase II, rhodamine channel. Arrowheads point to edges of the positive ceils. Bar, I0/~m. mutant, which did not contain a strong TGN localization determinant, was still internalized at 85 % the rate of ID5 (Fig. 7) . These experiments demonstrated that both YQRL-and YQDL-containing sequences can function as internalization determinants, even though only YQRZ-containing sequences seem to be effective for TGN localization.