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J. Biol. Chem., Vol. 279, Issue 52, 54046-54052, December 24, 2004

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Glyceraldehyde-3-phosphate Dehydrogenase Interacts with Rab2 and Plays an Essential Role in Endoplasmic Reticulum to Golgi Transport Exclusive of Its Glycolytic Activity^*

Ellen J. Tisdale, Carmen Kelly, and Cristina R. Artalejo

From the Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201

Rab2 requires atypical protein kinase C / (aPKC/) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC/ and binds to the aPKC/ regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20–50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC/ and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.

Received for publication, August 18, 2004 , and in revised form, September 27, 2004.

^* This work was supported by grants from the American Heart Association-Midwest Affiliate 0030385Z (to E. J. T.) and Public Health Service Grants DK58921 and MH47181 (to C. R. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, Wayne State University School of Medicine, 540 E. Canfield Ave., 6374 Scott Hall, Detroit, MI 48201. Tel.: 313-577-1007; Fax: 313-577-6739; E-mail: etisdale{at}med.wayne.edu.

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