1.3.32
VESICULAR TRAFFIC IN THE SECRETORY PATHWAY: CLATHRIN-COATED VESICLE ASSEMBLY AS A FUNGICIDE TARGET

J KEON and J HARGREAVES

IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS18 9AF, UK

Background and objectives
Clathrin (Cit)-coated vesicles are involved in the transport of proteins from either the plasma membrane or the trans-Golgi network (TGN) to specific compartments within the cell. In fungi, Cit-coated vesicles originating from the TGN are thought to play a role in the externalization of extracellular enzymes and in marshalling the components for cell wall assembly. Golgi- and plasma membrane-derived Cit-coated vesicles recruit different sets of tetrameric assembly proteins or adaptors. Adaptors of TGN-derived Cit-coated vesicles are composed of two adaptins (alpha-adaptin and gamma-adaptin) plus two smaller proteins; gamma-adaptin is specific to adaptors of CR-coated vesicles originating from the Golgi complex.

Results and conclusions
Growth and morphology of an Ustilago maydis osmotic remedial-thermosensitive (orts) mutant: an or-ts mutant of U. maydis was recovered after incubating UV-irradiated sporidia on agar medium at 25C. At 22C, growth of this mutant was similar to that of the wild-type. However, at 32C growth was severely impaired. Cells formed at the higher temperature were swollen and unable to develop by apical extension, and this form of growth led to the formation of rows of stunted, swollen cells. The mutant was able to grow at the restrictive temperature, albeit at a reduced rate, when the growth medium contained an osmotic stabilizer such as 1M sorbitol. However, cells grown under these conditions retained their abnormal morphology.

A U. maydis gamma-adaptin gene complements the or-ts mutant: a plasmid genomic library was transferred into the or-ts mutant and two plasmids containing overlapping DNA inserts were recovered from transformants that grew at the restrictive temperature. Analysis of the DNA sequence common to both inserts revealed a continuous open reading frame of 2652 bp encoding a protein containing 853 amino acids. Significant homology was found between the deduced amino acid sequence and the house mouse gamma-adaptin. Adaptors are known to consist of a central 'head' region flanked by two smaller appendages or 'ears', with each adaptin forming part of the 'head' with their N-terminal and an 'ear' with their C-terminal. Homology between the U. maydis gene product and the mouse gamma-adaptin was essentially restricted to the N-termini of the proteins.

Vesicle assembly as a fungicide target: the anilinopyrimidine fungicide, pyrimethanil, is known to act by interfering with the protein secretion pathway [1], thus demonstrating that vesicle transport from the TGN may be a potential target for new fungicide discovery. Furthermore, a fungal secondary metabolite, breveldin A, which also possesses antifungal activity, is known to prevent secretion of proteins by disrupting the structure and function of the Golgi complex. Indeed, an early event in the action of breveldin A is to prevent recruitment of gamma-adaptin and other vesicle coat proteins to the TGN [2].

References
1. Milling RJ, Richardson CJ, 1995. Pesticide Science 45, 43-48.
2. Robinson MS, Kreis TE, 1992. Cell 69, 129-138.