Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.4.4 (kinesin)
 5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-gamma-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin, KIFC2. Pre-incubation of vesicles with GTP-gamma-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-gamma-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.
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PMID:Regulation of early endocytic vesicle motility and fission in a reconstituted system. 1275 71

Kinesin-1 is a vesicle motor that can fold into a compact inhibited conformation that is produced by interaction of the heavy chain C-terminal tail region with the N-terminal motor domains (heads). Binding of the tail domains to the heads inhibits net microtubule-stimulated ATPase activity by blocking the ability of the heads to bind to microtubules with coupled acceleration of ADP release. We now show that folding of kinesin-1 also directly inhibits ADP release even in the absence of microtubules. With long heavy chain constructs such as DKH960 that exhibit a high degree of regulation by folding, the basal rate of ADP release is inhibited up to 30-fold compared to that of truncated DKH894 which has lost the inhibitory tail domains and does not fold. Inhibition of ADP release is also observed when separate head and tail domain constructs are mixed at low salt concentrations. This inhibition of ADP release by tail domains is formally analogous to the action of nucleotide dissociation inhibitors (NDI or GDI) for regulatory GTPases. In contrast to their inhibition of ADP release, tail domains accelerate the rate of ADP binding to nucleotide-free kinesin-1. Inhibition of release of ADP by tail domains is reversed by Unc-76 (FEZ1) which is a potential regulator of kinesin-1. Tail domains only weakly inhibit the initial slow release of Mg (2+) from the kinesin-MgADP complex but strongly inhibit the fast release of Mg (2+)-free ADP.
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PMID:Kinesin tail domains and Mg2+ directly inhibit release of ADP from head domains in the absence of microtubules. 1857 9