Figure 1.

Conserved WHXL motif is essential for plasma membrane association of Syt family. (A) Comparison of the C terminus of mouse Syts I–XI. Although the length of the C-terminal tail varies, the HWX(D/E)X₅RX₅WHXL sequence is highly conserved among mouse Syts I–XI. Arrowhead indicates the deletion point of SytΔC constructs. The WHXL motif is colored in red, and its mirror image (HW) is shown by yellow. (B) Plasma membrane association of Syt cytoplasmic domain. FLAG-tagged Syt-cytos (Left) or SytΔC-cytos (Right) was expressed in PC12 cells as described in Materials and Methods. PC12 cells were fixed, permeabilized, and stained with anti-FLAG (rabbit) antibody. Note that deletion of the WHXL motif of Syts I, III, IV, and VII completely prevented plasma membrane localization. Bar = 20 μm. (C) Amino acid sequence of mouse Syt-C and Syt-CM peptides and corresponding sequence of squid Syt I. Amino acid substitutions (WH to AA) are indicated by underlining. The Syt-C peptide sequence is highly conserved during evolution, and only one conserved amino acid substitution (Ile to Val) is found between mouse and squid Syt I. The Syt-C and Syt-CM peptides contain an artificial Cys residue at the N terminus.