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

• Published in: Microbiology (Society for General Microbiology) 2008-03, Vol.154 (3), p.842-851
• Main Authors: Moreno-Jimenez, Rocio, Garcia-Soto, Jesus, Martinez-Cadena, Guadalupe
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Blotting, Western; cdc42 GTP-Binding Protein - isolation & purification; Cell Fractionation; Centrifugation, Density Gradient; Cytoplasmic Vesicles - chemistry; Cytoplasmic Vesicles - enzymology; Fundamental and applied biological sciences. Psychology; Glucose Oxidase - analysis; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; GTP-Binding Proteins - analysis; Guanosine Triphosphate - metabolism; Isoelectric Point; Microbiology; Molecular Weight; Mucor - chemistry; Mucor - cytology; Mucor - enzymology; Mucor circinelloides; Mycology; Organelles; Phosphorus Radioisotopes - metabolism; rab GTP-Binding Proteins - isolation & purification; rhoA GTP-Binding Protein - isolation & purification; Zygomycetes
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2007/012179-0

Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apdo. postal 187, Guanajuato, Gto. 36000, Mexico Correspondence Guadalupe Martínez-Cadena margua{at}quijote.ugto.mx 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 [ - 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. Abbreviations: CHS, chitin synthase; GOX, glucose oxidase; G-protein, GTP-binding protein; IEF, isoelectron focusing