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First published online 20 May 2003
doi: 10.1242/jcs.00478
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Research Article |
1 Marion Bessin Liver Research Center
2 Department of Anatomy and Structural Biology, Albert Einstein College of
Medicine, Bronx, NY 10461, USA
* Author for correspondence (e-mail: wolkoff{at}aecom.yu.edu)
Accepted 14 March 2003
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-
-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--S, by contrast, repressed
motility. Addition of exogenous GST-Rab4- GTP--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.
Key words: Endocytosis, Microtubules, Rabs, Kinesin, Motility
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