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17
A potential role for Rab10 in ER network assembly
Amber English 1 , Gia Voeltz 1 .
University of Colorado, Boulder, CO 80303 1
Abstract:
The Endoplasmic Reticulum (ER) is a membrane bound organelle that includes the nuclear
envelope and the peripheral ER. The structure of the ER is quite dynamic; throughout the cell
cycle the ER membrane network is constantly rearranging in the cytoplasm using a fusion
mechanism that has not been characterized. We are interested in identifying the fusion
machinery required for ER network formation and maintenance. To identify the fusion
machinery, we are using an ER formation assay derived by fractionation of Xenopus laevis eggs.
In this system, ER vesicles fuse to form tubular networks in a GTP-dependent manner. To
isolate the GTP-dependent fusion machinery, we purified GTP binding proteins from the ER
extract on a GTP-agarose column and then identified the bound proteins by Mass Spectrometry.
Several Rab proteins were identified; Rab proteins are known GTPases that are involved in
membrane fusion of vesicles. Previous studies have shown that in vitro ER formation is
disrupted by the addition of a Rab GDP-dissociation inhibitor (GDI); indicating a Rab could be
involved in ER fusion. Of the several Rab GTPases identified only Rab8/10 has no previously
characterized function. We demonstrate that Rab 8/10 is tightly associated with the Xenopus
ER membrane vesicles but can be dissociated by the addition of recombinant RabGDI. Current
in vitro studies are focused on determining if Rab8/10 depletion from ER extracts affects ER
network formation. We find that a transient transfection of mCherry-Rab10 (human Rab8/10
homolog) into COS-7 cells co-localizes with an ER marker throughout the ER, consistent with a
potential role in ER fusion. Furthermore, transfection of a dominant negative form of Rab10,
mCherry-Rab10 (T23N), into COS-7 cells disrupts the ER in a manner consistent with a fusion
defect. Current studies are aimed at measuring the effects of Rab10 depletion and
overexpression on ER morphology to confirm a role for Rab10 in ER fusion.