Ras-related GTPases are important regulators of a wide variety of signaling pathways, including those required for organelle biogenesis and maintenance. Through their downstream effectors, members of the ARF and Rab sub-families control vesicle biogenesis and fusion, and they are recognized as central regulators of Golgi structure and function. We have been investigating two yeast ARF-like GTPases, Arl1p and ArI3p, that are highly conserved throughout eukaryotic evolution. Ar1p and ArI3p are localized to the Golgi apparatus where they are required for protein sorting and for Golgi-dependent uptake of iron via the high affinity uptake pathway. The functions of Arl1p and ArI3p are linked to signaling by the Ypt6p Golgi Rab GTPase: Ypt6p signaling is required for targeting and activation of ArI3p, and signaling by ArI3p is required for targeting and signaling by Arl1p. The goals of this research proposal are to identify factors that link signaling in this GTPase cascade and to elucidate the mechanisms by which ArI3p and Arl1p regulate Golgi function. ArI3p is unusual in that it is not myristoylated, but the N-terminal Methionine residue must be acetylated to be targeted to the Golgi.
In Specific Aim 1, we will test the hypothesis that the Golgi integral membrane protein, Sys1p, functions as a Golgi receptor for N-terminal acetylated Arl3p. The experiments proposed in Specific Aim 2 will identify the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that regulate Arl1p and ArI3p, and the effectors that link downstream signaling of Ypt6p and ArI3p to activation of ArI3p and Arl1 p, respectively. The defect the high affinity iron uptake system of ARL GTPase mutants is due to a failure to provide copper ion, a known co-factor for iron uptake, to the lumen of the Golgi. The experiments proposed in Specific Aim 3 will elucidate the role of Arl1 p and ArI3p in loading the Golgi with copper ion. Two human disorders, Wilson's and Menke's diseases, result from an inability of Golgi-localized copper efflux transporters and our research will contribute to an understanding how these proteins are localized and regulated within the cell.
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