Glyceraldehyde-3-phosphate dehydrogenase is required for vesicular transport in the early secretory pathway.
Protein transport in the early secretory pathway requires Rab2 GTPase. This protein promotes the recruitment of soluble components that participate in protein sorting and recycling from pre-Golgi intermediates (vesicular tubular clusters (VTCs)). We previously reported that a constitutively activated form of Rab2 (Q65L) as well as Rab2 wild type promoted vesicle formation from VTCs. These vesicles contained Rab2, beta-COP, p53/gp58, and protein kinase Ciota/lambda but lacked anterograde-directed cargo. To identify other candidate Rab2 effectors, the polypeptide composition of the vesicles was further analyzed. We found that vesicles released in response to Rab2 also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To study the relationship of this enzyme to Rab2 function, we performed a quantitative binding assay to measure recruitment of GAPDH to membrane when incubated with Rab2. Rab2-treated microsomes showed a 5-10-fold increase in the level of membrane-associated GAPDH. We generated an affinity-purified anti-GAPDH polyclonal to study the biochemical role of GAPDH in the early secretory pathway. The antibody arrests transport of a reporter molecule in an assay that reconstitutes ER to Golgi traffic. Furthermore, the affinity-purified antibody blocked the ability of Rab2 to recruit GAPDH to membrane. However, the antibody did not interfere with Rab2 stimulated vesicle release. These data suggest that GAPDH is required for ER to Golgi transport. We propose that membranes incubated with anti-GAPDH and Rab2 form "dead end" vesicles that are unable to transport and fuse with the acceptor compartment.
Tisdale
E J
EJ
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. etisdale@med.wayne.edu
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D020756:Coatomer Protein / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D020693:rab2 GTP-Binding Protein / Q000378:metabolism
D020756:Coatomer Protein; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D020693:rab2 GTP-Binding Protein