Abstract
Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Ciota (aPKCiota) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCiota in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCiota were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCiota. Because GAPDH binds to the carboxyl terminus of alpha-tubulin, we characterized the distribution of tyrosinated/detyrosinated alpha-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated alpha-tubulin; however, aPKCiota was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCiota-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.
Highlights
Pleomorphic structures that sort anterograde-directed cargo from recycling proteins and trafficking machinery retrieved to the endoplasmic reticulum (ER) [3,4,5,6]
We have previously reported that glyceraldehyde-3phosphate dehydrogenase (GAPDH) and atypical PKC are Rab2 effectors that interact directly with the Rab2 amino terminus and with each other [8, 9]
Rab2 Requires GAPDH and aPKC to Associate with MTs— To learn if Rab2 binds directly to MTs, an in vitro MT binding assay was performed in which purified recombinant Rab2 was incubated with paclitaxel assembled MTs for 30 min at 37 °C
Summary
Quantitative Membrane Binding Assay—HeLa membranes were prepared as described previously [10]. Purified recombinant Rab, aPKC, and aPKC (K274W) [7, 36] or purified recombinant Rab amino-terminal fragment, prepared as described below, was added at the concentrations indicated under “Results,” and the reaction mix was incubated on ice for 10 min. The cells were incubated for 30 min with either anti-Tyr-tubulin (Millipore), or an affinity-purified Rab polyclonal antibody [35], or an affinity-purified polyclonal to p53/p58 [37], or anti-GAPDH (Cell Signaling, Danvers, MA), anti-aPKC (GenScript Corp., Piscataway, NJ), or fluorescein isothiocyanate-conjugated anti-Xpress (Invitrogen); washed extensively with PBS; incubated with Alexa Fluor 488 chicken antimouse antibody and Alex Fluor 594 chicken anti-rabbit; washed extensively with PBS; mounted in Mowiol containing 1,4-diazabicyclo[2.2.2]octane (Sigma); and viewed with a Zeiss AxioImager epifluorescence microscope (Carl Zeiss, Gottingen, Germany) and photographed with an AxioCamMRm camera (Zeiss Microimaging, Thornwood, NY) using AxioVision Z-stack software to capture 20 images of ϳ3.6 m (Zeiss Microimaging). The Rab amino-terminal deletion mutant was generated, as outlined previously [9]
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