CHARACTERIZATION OF THE ARF FAMILY OF REGULATORY GTP-BINDING PROTEINS

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This section is involved in studies of the structure, function and mechanism of action of members of the superfamily of regulatory GTP binding proteins; particularly the members of the ADP-ribosylation factor (ARF) family. ARF has been shown to have essential functions in the maintenance of the integrity and proper functioning of the endoplasmic reticulum, Golgi complex, and endosome compartments. ARF proteins also stimulate phospholipase D, likely serving as the direct activators of this effector. The cloning and sequencing of novel members of the ARF family in humans and the yeast, S. cerevisiae, continues. There are currently 6 mammalian ARF proteins and 6 mammalian ARF-like proteins fully cloned and sequenced. In addition, we have partial sequence of at least five more members in yeast, and several more in humans. Yeast genetics continues to provide new clues to the actions of ARF proteins in eukaryotes. A conditional lethal allele of ARF1 revealed a role for ARF in sporulation. Analysis of another allele of arf1 led to the observation that ARF function is also required for respiration. It is likely that the roles for ARF proteins in these essential cellular processes provide important clues toward understanding the mechanisms of action of these critical regulatory proteins. The two-hybrid technique was used to identify other components in an ARF signaling pathway. This approach led to the cloning of 6 human genes whose products interact with human ARF3. Northern blots revealed that five of these 6 genes are expressed in a wide variety of human tissues, and one with a more limited tissue distribution. The three dimensional crystal structure of ARF~GDP has been solved. Work continues on the active, ARF~GTP and myristoylated ARF structures as each should aid in the determination of critical functional domains and design of specific inhibitors.