Abstract
We recently have identified a new cytoplasmic linker protein (CLIP), CLIPR-59, which is involved in the regulation of early endosome/trans-Golgi network dynamics. In contrast with CLIP-170, CLIPR-59 is not localized to microtubules at steady state but is associated with the trans-Golgi network and the plasma membrane. Here we show that the last 30 amino acids (C30) are sufficient for membrane targeting and that two cysteines in the C30 domain are palmitoylated. We demonstrate that CLIPR-59 is associated with lipid rafts via its C-terminal palmitoylated domain. In vitro experiments suggest that CLIPR-59 and its microtubule-binding domain alone have a better affinity for unpolymerized tubulin or small oligomers than for microtubules. In contrast with the CLIP-170 microtubule-binding domain, the CLIPR-59 microtubule-binding domain diminishes microtubule regrowth after nocodazole washout in vivo, showing that this domain can prevent microtubule polymerization. In contrast with the role of linker between membranes and microtubules that was proposed for CLIP function, CLIPR-59 thus may have an "anti-CLIP" function by preventing microtubule-raft interactions.
Highlights
The microtubule network is required for intracellular organization and movement
In cells expressing a higher amount of GFP-C30 or GFP-CLIPR-59 full-length protein (GFP-C59) proteins, the plasma membrane was labeled indicating that CLIPR-59 distribution was not restricted to but concentrated on Golgi membranes (Fig. 1A, arrowhead)
CLIPR-59 Is Targeted to Lipid Raft by Tandem Palmitoylations—We have characterized the mechanism of CLIPR-59 membrane targeting and demonstrated that CLIPR-59 is a raft-associated cytoplasmic linker protein (CLIP) endowed with an unusual inhibition of microtubule regrowth activity
Summary
The microtubule network is required for intracellular organization and movement. The dynamic instability of microtubules allows a rapid exploration of the cytoplasmic space, and the intrinsic asymmetry of the tubulin polymer is used by molecular motors to establish intracellular polarity. We found that the last 30 amino acids of CLIPR-59 are necessary and sufficient to target it to the TGN and to the plasma membrane and that a double palmitoylation on tandem cysteines within this domain is responsible for the raft targeting. Immunofluorescence experiments performed on GFP-C30 transiently transfected HeLa cells showed that the GFP-C30 protein was addressed to membranes and co-localized with the cis-Golgi marker GM-130 (Fig. 1A).
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