Small GTP-binding proteins are associated with chitosomes and vesicles carrying glucose oxidase from Mucor circinelloides

Abstract

Fractions enriched with chitosomes and vesicles carrying glucose oxidase (GOX) activity from the dimorphic zygomycete Mucor circinelloides were obtained using two successive sucrose gradients, the first a linear-log and the second an isopycnic gradient. Using an [alpha-(32)P]GTP-binding assay, we detected the association of small GTP-binding proteins (21 and 17 kDa) with both types of vesicles. In addition, by ADP-ribosylation with C3 exotoxin, and Western blot analysis with specific antibodies, we identified the small GTPases RhoA (Rho1p) and Rab8, and a 17 kDa protein, with pI values of 6.0, 6.1, and 6.2 and molecular masses of 21, 21 and 17 kDa, respectively, associated with those vesicles carrying GOX activity. Rab and Cdc42 proteins with pI values of 6.1 and 6.2 and molecular masses of 21 and 17 kDa, respectively, were found associated with chitosomes. These data indicate the presence in M. circinelloides of low molecular mass G-proteins in chitosomes and in vesicles carrying GOX activity. The difference in association of Rho1 and Cdc42, with vesicles carrying GOX activity and chitosomes, respectively, indicates that each of these proteins probably controls formation, transport and specific plasma membrane site docking of the respective vesicles.