Characterization of Rah, a low molecular weight GTP -binding protein
Abstract
Low molecular weight GTP binding proteins, better known as small GTPases, are a large superfamily of proteins which control diverse cellular events including cell growth, transformation, cytoskeletal Changes, vesicular trafficking, and nuclear transport. On the basis of differences in structure and function, the small GTPases are divided into the Ras, Rac/Rho, Rab/Arf, and the Ran families. The Rab small GTPases are regulators of vesicle trafficking pathways in the cell. In a search for Rab-like small GTPases, the Rah cDNA was cloned from the HT4 mouse neural cell line. The research and data presented here are centered on the molecular, biochemical and cellular characterization of this Rah gene and the Rah protein it encodes. The full-length open reading frame of the mouse Rah cDNA encodes a protein of 28.8 kDa. Sequence alignment and analysis suggest that Rah is a previously unknown member of the Rab family. Unlike other small GTPases, Rah has a unique N-terminal extension of 51 amino acids and a novel C-terminal prenylation motif, -CCP. Recombinant GST-tagged Rah protein was expressed in E. coli. Biochemical analysis revealed that Rah protein has the ability to bind GTPγS and can hydrolyze GTP. Site directed mutants of Rah, RahT84N and RahQ111R were produced, which on the basis of previous studies were predicted to be locked in GDP-bound and GTP-bound conformations, respectively. The RahT84N mutant had lower affinity for GTPγS and was insensitive to the Mg2+ concentration for nucleotide exchange. Immunoblot analysis revealed that Rah protein is expressed in heart, liver, kidney and skeletal muscle. Immunofluorescence microscopy suggested that Rah co-localizes to the N-acetyl glucosamine transferase containing Golgi compartments. The overexpression of Rah and Rah mutants in HT4 cells did not result in any dramatic effect on Golgi morphology. The overexpression of Rah and Rah mutants along with vesicular stomatitis virus glycoprotein (VSV-G) caused the VSV-G to accumulate in the Golgi complex, presumably because it was unable to exit the Golgi complex. The results of this study indicate that Rah is a new member of the Rab family and suggest that Rah has a specialized sorting function in the Golgi complex of certain tissues.
Degree
Ph.D.
Advisors
Asai, Purdue University.
Subject Area
Cellular biology|Molecular biology
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