Moreover, live imaging of Ibrutinib purchase GFP-tagged Myr-GRIP1b revealed that a subset of these recycling endosomes is highly motile (Movie S1). Together, these data suggest that mimicking N-terminal palmitoylation targets GRIP1b to motile dendritic trafficking endosomes. These findings suggested that wild-type GRIP1b might distribute between the diffuse pattern of GRIP1b-C11S and the punctate pattern of Myr-GRIP1b. However, dendritic puncta of transfected GRIP1bwt were far less

numerous than those seen with Myr-GRIP1b (Figures 3D and 3F). We hypothesized that this might be due to limiting endogenous DHHC5 PAT activity. Consistent with this notion, transfection of wild-type DHHC5 transformed GRIP1bwt distribution in two ways. First, DHHC5wt increased the level of GRIP1bwt detected in distal dendrites (Figure 4A; quantitated in Figure 4B). Second, DHHC5wt transformed GRIP1bwt staining from a largely diffuse pattern to one that was strikingly FK228 punctate (Figure 4A; quantitated in Figure 4C). Indeed, the number of GRIP1bwt puncta in distal dendrites of HA-DHHC5 expressing neurons approached that seen with Myr-GRIP1b. Changes in GRIP1bwt distribution were likely due to direct palmitoylation because HA-DHHC5wt expression did not affect GRIP1b-C11S distribution.

Strikingly, neither DHHS5 nor DHHC5ΔC increased GRIP1b targeting to dendrites (Figures 4A–4C), despite the normal dendritic targeting of these mutants (Figure S4). Together,

these results suggest that palmitoylation by DHHC5 targets GRIP1b to recycling endosomes and that, as in heterologous cells (Figure 1G), this phenotypic effect requires both the PAT activity and PDZ binding ability of DHHC5. The rapid turnover of palmitate on GRIP1 suggested that GRIP1 vesicular localization might be affected by acute inhibition of palmitoylation. Indeed, acute treatment (90 min) with 2-Bromopalmitate dramatically dispersed GRIP1 puncta in both proximal and distal dendrites (Figure 5A). These findings are consistent Farnesyltransferase with palmitoylation reversibly targeting GRIP1b to dendritic endosomes and suggested that palmitoylation might modulate interactions with other GRIP1 partners that control vesicle trafficking. One such trafficking protein is the dendritic kinesin motor protein KIF5, whose interaction with GRIP1 is critical for GluA2 trafficking within dendrites (Setou et al., 2002). We, therefore, addressed whether GRIP1 palmitoylation might modulate GRIP1 interactions with KIF5, by coexpressing KIF5C with wild-type, nonpalmitoylatable, or Myr-GRIP1b in heterologous cells. Strikingly, myristoylated GRIP1 bound more KIF5C than did wild-type GRIP1, while GRIP1b-C11S bound KIF5C only minimally (Figure 5B). Moreover, in neurons a Myr-GRIP1b mutant lacking the previously reported KIF5-binding domain of GRIP1 (Setou et al., 2002; myr-GRIP1b-delKBD) showed markedly reduced targeting to distal dendrites (Figures 5C and 5D).