GTP-binding proteins are molecular switches that regulate a host of cellular processes including cell growth and division. One class of small, monomeric GTP-binding proteins, the ARF family, plays an essential role in the organization and movement of a wide array of intracellular membranes in all eukaryotic cells. Newly synthesized proteins destined for export must pass from the endoplasmic reticulum through the various compartments of the Golgi, trans-Golgi network (TEN) and secretory vesicles; this transport is accomplished by a vectorial series of vesicle fusions. Vesicles bud off the host membrane and then fuse selectively with the appropriate target membrane. Budding requires the binding of a high molecular weight protein complex (coatomer) to the membrane. And coatomer binding requires prior binding of ARF. ARF proteins (ADP-ribosylation factor; required for cholera toxin dependent ADP-ribosylation of the regulatory component of adenylate cyclase, Gs) are ubiquitous in eukaryotes. The ARF1 gene encodes a highly conserved protein of 181 amino acids in mammals and in the yeast S. cerevisiae. Variants are found in all eukaryotes examined thus far, including plants and insects. The function of ARF has been conserved between yeast and man. Deletion of the two ARF genes in yeast is lethal. Myristoylation of the amino terminus of ARF is essential for functions in vivo. A number of in vitro assays of ARF functions have been described, most related to activities as a regulator of intracellular vesicle movement (e.g. budding and fusion). ARF has been shown to regulate multiple steps in both the exocytic and endocytic pathways. In addition to regulation of ER-Golgi, intra-Golgi transport and endosome fusion, ARF proteins have also been implicated in nuclear vesicle fusion, coatomer recruitment onto ER and Golgi membranes and as the direct regulator of the intrinsic membrane effector, phospholipase D. Further, the inhibition of activation of ARF has been shown to be a critical component in the actions of both Brefeldin A and ilimaquinone; each is currently being evaluated at NCI as a novel anti-tumor agent. The ARF cloned from Giardia lamblia is sufficiently different from mammalian ARF that the design of specific inhibitors of this essential protein can be envisaged as a potential anti- parasitic agent. A better understanding of the structure and functional domains of this essential regulatory molecule would greatly assist both basic cell biology studies and the design of selective inhibitors of critical cellular processes for potential chemotherapeutic advantage. Recombinant human ARF1 with GDP bound has been crystallized in a form diffracting to better than 2A resolution. The crystals are monoclinic, space-group C2 (a=122.8, b=45.8, c=89.3, beta=131.l ), with two ARF molecules in the assymetric unit.
The aim of this proposal is to obtain the structures of ARF1 in its GDP-bound and GTP-bound states by X-ray crystallography. An interpretable electron density map of ARF1-GDP has been calculated at 2A resolution. The structures of the myristoylated enzyme in both states will also be determined.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050430-03
Application #
2188280
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1994-09-22
Project End
1998-08-31
Budget Start
1996-09-01
Budget End
1998-08-31
Support Year
3
Fiscal Year
1996
Total Cost
Indirect Cost
Name
Brandeis University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454