This project is focussed on the elucidation of structures of proteins in the secretory pathway. The key protein in vesicle formation is the ADP ribosylation factor that sequesters the proteins required for vesicle formation to the membrane. The protein behaves like an on/off switch where the GDP form is soluble in the cytosol and the GTP form is membrane associated. One of the elements that is essential for membrane association is a myristyl group at the N-terminus of the protein, without which membrane association does not occur. So far, the structure of the human ARF1 has been determined, and structures of yeast ARF1 and ARF2 are underway. In addition to the ARF's, cells also contain ARF-related proteins, called ARL's, that seem to have a function similar to that of ARF, but not identical. We have crystallized ARL1 from drosophila in hopes of obtaining a structure that might indicate what the differences between ARL and ARF are, and how they relate to cellular function. These crystals are too small for in-house data collection. During the trip to CHESS (8.-11. April 1998) a number of ARL crystals were screened, however, none of them diffracted to high enough resolution (better than 8A) for useful data collection. The enzyme responsible for myristylation at the N-terminus of the ARF's and ARL's is N-myristyl transferase. The structure of this enzyme from yeast has been determined, but is sufficiently different from the human enzyme by sequence comparison that we are determining the structure of the human enzyme. The protein crystallizes slowly and produces very small crystals that do not diffract well enough for in-house data collection. A 85% complete data set (to 3A resolution) of the hNMT was obtained at CHESS (8.-11. April 1998) under cryo conditions. We are in the process of using the yeast NMT coordinates to these data in molecular replacement attempts. So far, we have obtained two promising solutions that have to be analyzed further before we have a definitive answer.
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