Dataset Information

The C. elegans rab family: identification, classification and toolkit construction.

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).

SUBMITTER: Gallegos ME

PROVIDER: S-EPMC3504004 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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The C. elegans rab family: identification, classification and toolkit construction.

Gallegos Maria E ME Balakrishnan Sanjeev S Chandramouli Priya P Arora Shaily S Azameera Aruna A Babushekar Anitha A Bargoma Emilee E Bokhari Abdulmalik A Chava Siva Kumari SK Das Pranti P Desai Meetali M Decena Darlene D Saramma Sonia Dev Devadas SD Dey Bodhidipra B Doss Anna-Louise AL Gor Nilang N Gudiputi Lakshmi L Guo Chunyuan C Hande Sonali S Jensen Megan M Jones Samantha S Jones Norman N Jorgens Danielle D Karamchedu Padma P Kamrani Kambiz K Kolora Lakshmi Divya LD Kristensen Line L Kwan Kelly K Lau Henry H Maharaj Pranesh P Mander Navneet N Mangipudi Kalyani K Menakuru Himabindu H Mody Vaishali V Mohanty Sandeepa S Mukkamala Sridevi S Mundra Sheena A SA Nagaraju Sudharani S Narayanaswamy Rajhalutshimi R Ndungu-Case Catherine C Noorbakhsh Mersedeh M Patel Jigna J Patel Puja P Pendem Swetha Vandana SV Ponakala Anusha A Rath Madhusikta M Robles Michael C MC Rokkam Deepti D Roth Caroline C Sasidharan Preeti P Shah Sapana S Tandon Shweta S Suprai Jagdip J Truong Tina Quynh Nhu TQ Uthayaruban Rubatharshini R Varma Ajitha A Ved Urvi U Wang Zeran Z Yu Zhe Z

PloS one 20121121 11

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 tow ...[more]

PMID: 23185324

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Project description:BackgroundAntibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms.Methodology/principal findingsWe report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1), a component of synaptic vesicles; to Rim (UNC-10), a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1), a component of centrosomes; to CENP-C (HCP-4), which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2), a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5); to the nuclear envelope protein lamin (LMN-1); to EHD1 (RME-1) a marker for recycling endosomes; to caveolin (CAV-1), a marker for caveolae; to the cytochrome P450 (CYP-33E1), a resident of the endoplasmic reticulum; to beta-1,3-glucuronyltransferase (SQV-8) that labels the Golgi; to a chaperonin (HSP-60) targeted to mitochondria; to LAMP (LMP-1), a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7) of the 26S proteasome; to dynamin (DYN-1) and to the alpha-subunit of the adaptor complex 2 (APA-2) as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1) and cadherin (HMR-1), both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1), which localized to apical membranes; to an ERBIN family protein (LET-413) which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7) which localizes to the plasma membrane at cell-cell contacts. In addition to working in whole mount immunocytochemistry, most of these antibodies work on western blots and thus should be of use for biochemical fractionation studies.Conclusions/significanceWe have produced a set of monoclonal antibodies to subcellular components of the nematode C. elegans for the research community. These reagents are being made available through the Developmental Studies Hybridoma Bank (DSHB).

Dataset Information

The C. elegans rab family: identification, classification and toolkit construction.

Publications

The C. elegans rab family: identification, classification and toolkit construction.

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