title: Rab2 utilizes glyceraldehyde-3-phosphate dehydrogenase and protein kinase C{iota} to associate with microtubules and to recruit dynein.
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.
authors.affiliation: Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. etisdale@med.wayne.edu
authors.forename: Ellen J
authors.initials: EJ
authors.lastname: Tisdale
authors.forename: Fouad
authors.initials: F
authors.lastname: Azizi
authors.forename: Cristina R
authors.initials: CR
authors.lastname: Artalejo
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D055571:Secretory Pathway; D014404:Tubulin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D020693:rab2 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D000099293:Protein Kinase C-lambda
chemical_list: D004398:Dyneins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007527:Isoenzymes; D011493:Protein Kinase C; D014404:Tubulin; D014443:Tyrosine; D020693:rab2 GTP-Binding Protein; D000099293:Protein Kinase C-lambda