Endocytosis efficiency of VAMPs 2, 3, and 8.

<p>a, VAMPs 2, 3, and 8 have a similar domain structure, consisting of a short, apparently unstructured N-terminal domain, a SNARE domain, a transmembrane domain (TMD), and a very short lumenal/extracellular domain, to which a double HA (hemagglutinin) tag was added when making the constructs. The CALM-binding domain and the positions of the residues that were mutated are indicated for VAMP8. b, The FACS-based assay described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064514#pone-0064514-g001" target="_blank">Figure 1</a> shows that endocytosis efficiency is highest for VAMP8, and is strongly reduced when CALM is depleted. The VAMP8 P3P4 mutant, which is not impaired in CALM binding, is endocytosed as efficiently as the wild-type construct, and both are strongly affected by CALM knockdown. In contrast, the P1P2 and AA mutants, which are unable to bind CALM, are poorly endocytosed, and the CALM knockdown has no effect on the endocytosis efficiency of these constructs. c, Western blot showing steady state levels of wild-type and mutant VAMP8 in three different cell lines for each construct. Cell lysates were adjusted for equal protein concentration. Although there is some variability from one line to another, the two non-CALM-binding mutants are always expressed at much higher levels than wild-type VAMP8 or the P3P4 mutant. d, The differences in expression are due to differences in degradation, not in synthesis. When de novo protein synthesis was blocked with cycloheximide, the levels of the two CALM-binding constructs decreased rapidly over four hours, while the levels of the two non-CALM-binding mutants were not appreciably affected.