TRANSPORT OF THE HYDROPHOBIN CRYPARIN THROUGH THE SECRETORY PATHWAY OF CRYPHONECTRIA PARASITICA
PM McCABE and NK VAN ALFEN
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, 77843-2132, USA
Background and objectives
The virus containing strains of the chestnut blight fungus, Cryphonectria parasitica, show standard growth in culture but the virus impairs normal development. This developmental deficiency is manifested as a reduction of pigmentation, conidial formation, sexual reproduction and virulence. Strains show no cytopathy as a result of virus infection, the only obvious difference being that the virus-infected strains accumulate small membrane vesicles. These vesicles are also present in uninfected strains, although at a concentration of about six-fold less than in infected strains. These vesicles were isolated by a process of purification of both the virus genomic dsRNA and an associated RNA polymerase activity . A number of genes have been shown to be differently regulated as a result of viral infection. The ones studied in this laboratory are all extracellular proteins that are down-regulated by the virus. Included are an extracellular laccase, a hydrophobin called cryparin , and a mating type-specific pheromone, MF1-1. All of these contain signal sequences that are followed by a pro region which in turn is cleaved after a pair of dibasic residues, presumably by a protease similar to the yeast serine protease, Kex2p. In yeast all Kex2p-processed proteins are transported through the cell in the same secretory vesicle fraction. These observations led us to hypothesize that the virus is utilizing a host secretory system for replication, resulting in an increase in dysfunctional vesicles, and a decrease in transport of the normal cargo proteins. This hypothesis is being tested by examination of cryparin transport.
Results and conclusions
Antibody to cryparin identified the protein in the virus-containing vesicle fraction. Associated with the vesicles were two forms of cryparin, the expected 21-kDa molecular-weight form, and a second higher molecular-weight protein (36 kDa). In culture supernatant only the mature form of the protein was seen, indicating that cryparin must undergo processing as it is exported. The 36-kDa form was present in much lower quantities but was far more antigenic, suggesting it is glycosylated. To test this, cryparin was extracted from cells and purified by HPLC. Using the 'Glycotrack' carbohydrate detection kit, the 36-kDa protein was shown to be glycosylated. Chemical deglycosylation using the 'Glycofree' carbohydrate removal kit resulted in a shift in weight of the 36-kDa protein to 21 kDa, the size of the mature cryparin molecule. These results were confirmed using a cryparin-specific antibody.
Transport of cryparin through the cell to the culture fluid was followed using 35S-labelled cysteine which was incorporated into the cryparin protein during translation. Aliquots of cells were removed at 5 min time intervals, and cryparin extracted from both the cellular and supernatant fractions and detected by autoradiography following SDS-PAGE. Cryparin could be detected within the cells as quickly as 5 min after labelling, and the mature form detected in the culture supernatant 30 min after labelling, showing very efficient transport of the protein.
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