Abstract
Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).
Highlights
The Rab Family of Monomeric GTPases The RAS superfamily of monomeric GTPases is widely conserved and includes five main families: Ras, Rho, Arf, Ran and Rab
With the multiple sequence alignment (MSA) of the expanded list, we calculated a single Rab Family (RabF) percent identity score based on conservation with the consensus sequences for RabF1 through RabF5 combined (27 amino acids in total)
Consistent with recent results [33], we expand the size of the C. elegans Rab family by three to 31 members
Summary
The Rab Family of Monomeric GTPases The RAS superfamily of monomeric GTPases is widely conserved and includes five main families: Ras, Rho, Arf, Ran and Rab. Rab localizes to clathrin-coated vesicles budding from the plasma membrane, early endosomes and transport vesicles in between. These observations reflect Rab5’s specific role in clathrin uncoating and target selection of vesicles travelling from the plasma membrane to the early endosome. All aspects of vesicular trafficking including intracellular transport of proteins, ligand secretion, receptor trafficking and protein degradation is fundamental to the function of the eukaryotic cell. A number of bacterial pathogens express regulators of specific host Rabs to facilitate cell entry and avoid degradation by the lysosome, thereby promoting the process of infection [3]
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