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

To identify specific amino acid residues critical for signaling localization to the TGN, 10 of the 11 COOH-terminal residues of the ID5 chimeric protein were individually mutated to alanine residues (Fig. 6, top) . The immunofluorescence microscopy pattern of each mutant was determined in transiently transfected NRK cells. Substitution of an alanine residue for either Y333 or L336 resulted in loss of TGN localization and a concomitant increase in plasma membrane labeling (Fig. 6) ; the remaining alanine mutations had no effect on the TGN localization of the chimeric protein (Fig. 6 , and data not shown). Interestingly, whereas replacement of R335 by an alanine residue did not affect the distribution of the protein, replacement by an aspartate residue caused decreased intensity of TGN staining and increased expression at the plasma membrane and in cytoplasmic vesicles (Fig. 6) . Thus, these results indicated that Y333 and L336 are critical for TGN localization of the chimeric molecules. R335, on the other hand, can be replaced by an alanine but not an aspartic acid residue for efficient TGN localization.