Members of the ARF family of regulatory GTPases function as nodes in cell signaling to coordinate essential cell processes; including membrane traffic, energy metabolism, and the cytoskeleton. The focus of this application is one of those GTPases, ARL2, and its binding partners. An understanding of the molecular mechanisms of ARL2 action to carry out or regulate essential cell processes will reveal novel insights into fundamental aspects of cell biology as well as providing potential targets for intervention to alte the course of human diseases; including but not limited to cancer, heart disease, neurodegeneration, retinal degeneration, and deafness. During the previous four years of funding we have provided rigorous tests of several models for ARL2 actions in different parts of eukaryotic cells, discarding some and refining others. We have also generated many key reagents, including the first purified protein preparations of mg levels of four different proteins and a series of point mutations in ARL2 that allow dissection of its different essential roles in eukaryotic cells. Thus, we are poised to make much more rapid progress in our understanding of the molecular mechanisms of regulation of tubulin folding and polymerization, ATP generation in mitochondria, and mitochondrial fission and motility. In the next funding period we propose three new, specific aims that are logical extensions of our previous work but which promise to have a far broader impact on several fields of cell signaling and regulation.
In aim 1 we will test the model that ARL2 acts with the tubulin-specific co-chaperone cofactor D (TBCD) to regulate tubulin folding, microtubule density or polymerization, and less directly cell division.
In aim 2 w will test the hypothesis that ARL2 regulates ATP production in mitochondria via changes in cristae remodeling and mitochondrial fusion. And in aim 3 we examine how mitochondrial fusion and motility are regulated by the ARL2 effector and GAP, ELMOD2.

Public Health Relevance

Essential cellular processes common to all cells are often controlled by GTPases within the RAS superfamily and ARF family and dysfunction in those regulatory systems result in human diseases ranging from cancer, heart disease, neurodegeneration, retinal degeneration, and deafness. One of these GTPases, ARL2, and its binding partners has been linked to each of these diseases. We propose basic studies to determine the mechanism by which ARL2 works with cofactor D (TBCD) in cytosol to regulate tubulin folding and microtubules, and in mitochondria to regulate fusion, cristae remodeling and ATP levels, a subset of which are mediated by the ARL2 GAP, ELMOD2.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM090158-05A1
Application #
8964313
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Anderson, Vernon
Project Start
2010-09-01
Project End
2019-04-30
Budget Start
2015-07-01
Budget End
2016-04-30
Support Year
5
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Emory University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Newman, Laura E; Schiavon, Cara R; Turn, Rachel E et al. (2017) The ARL2 GTPase regulates mitochondrial fusion from the intermembrane space. Cell Logist 7:e1340104
Francis, Joshua W; Newman, Laura E; Cunningham, Leslie A et al. (2017) A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and ?-Tubulin Is Required for Maintaining the Microtubule Network. J Biol Chem 292:4336-4349
Francis, Joshua W; Goswami, Devrishi; Novick, Scott J et al. (2017) Nucleotide Binding to ARL2 in the TBCD?ARL2??-Tubulin Complex Drives Conformational Changes in ?-Tubulin. J Mol Biol 429:3696-3716
Bhatt, Jay M; Viktorova, Ekaterina G; Busby, Theodore et al. (2016) Oligomerization of the Sec7 domain Arf guanine nucleotide exchange factor GBF1 is dispensable for Golgi localization and function but regulates degradation. Am J Physiol Cell Physiol 310:C456-69
Wigington, Callie P; Morris, Kevin J; Newman, Laura E et al. (2016) The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA. J Biol Chem 291:22442-22459
Seixas, CecĂ­lia; Choi, Soo Young; Polgar, Noemi et al. (2016) Arl13b and the exocyst interact synergistically in ciliogenesis. Mol Biol Cell 27:308-20
Flex, Elisabetta; Niceta, Marcello; Cecchetti, Serena et al. (2016) Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy. Am J Hum Genet 99:962-973
Francis, Joshua W; Turn, Rachel E; Newman, Laura E et al. (2016) Higher order signaling: ARL2 as regulator of both mitochondrial fusion and microtubule dynamics allows integration of 2 essential cell functions. Small GTPases 7:188-196
Newman, Laura E; Schiavon, Cara; Kahn, Richard A (2016) Plasmids for variable expression of proteins targeted to the mitochondrial matrix or intermembrane space. Cell Logist 6:e1247939
Kahn, Richard A; Lambright, David G (2015) A PH Domain with Dual Phospholipid Binding Sites Regulates the ARF GAP, ASAP1. Structure 23:1971-3

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