It has been shown that the membrane protein podocalyxin (PCX)/Gp135 localizes to microvilli in part through its C-terminal tail binding to the PDZ2 domain of EBP50 ( Yu et al., 2007 Francis et al., 2011). This is very unexpected, as EBP50 binds active ezrin with a K d in the nanomolar range, so it would be expected to have a very slow off-rate and therefore show similar dynamics to ezrin. Surprisingly, GFP-EBP50 recovered very fast, within 5 s, showing that it is a highly dynamic component of microvilli ( Fig.
#Single site mutant protein turnover full
As TROP2 is a transmembrane protein, its recovery is restricted to two dimensions, suggesting that ezrin is relatively firmly bound in the microvillus, as it has full access to the cytoplasm, which is consistent with our model. 1, B and C), which is consistent with what has been seen for ezrin by two-photon microscopy ( Coscoy et al., 2002). Both TROP2-GFP and ezrin-GFP recover slowly, with about half the microvillar fluorescence returning in 30 s ( Fig. 1, A and C), which fit well to double exponential curves (Fig. Experiments on multiple cells showed very reproducible recovery data ( Fig. For these experiments, each construct was expressed at a low level in JEG-3 cells and a small area bleached, and the recovery was then monitored. We first determined the dynamics of the microvillar transmembrane protein TROP2-GFP, which binds to ezrin (unpublished data) and ezrin-GFP. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins. Using a novel in vitro photoactivation fluorescence assay, the EBP50–ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. However, EBP50 was highly dynamic, turning over within seconds. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton.
0 kommentar(er)
