Rho1 and other GTP-binding proteins are associated with vesicles carrying glucose oxidase activity from Fusarium oxysporum f. sp. lycopersici

Abstract

In eucaryotic cells, the delivery of a secreted protein to the plasma membrane via vesicles must include transport, recognition, and fusion events. Proteins exposed on the cytoplasmic face of the secretory vesicles play a role in these events; these include the GTP-binding proteins, which are crucial components in this process. Fractions enriched with vesicles carrying glucose oxidase (GOX) activity from Fusarium oxysporum f. sp. lycopersici, a soilborne fungal pathogen causing vascular wilt on tomato plants, were obtained using two successive sucrose gradients, the first a linear-log and the second an isopycnic gradient. In this study, we used the following Fusarium strains: a wild-type and a strain carrying a Δrho1 loss-of-function mutation (presenting dramatically reduced virulence). By ADP-ribosylation with C3 exotoxin, and Western blot analysis with specific antibodies, we identified the small GTPases Rho1, Rho4, Cdc42 and Rab8, and a heterotrimeric Gα protein associated with vesicles carrying GOX activity. This was done for both strains, with the exception of Rho1, which was absent in the mutant strain; in addition, the levels of the Cdc42 protein were observed to be higher in the Δrho1 strain. These data indicate that three Rho proteins, Rho1, Rho4, and Cdc42, are present in secretory vesicles carrying GOX activity in F. oxysporum, and that Rho1 is not essential for the transport and secretion of, at least, cargo proteins carried in secretory vesicles, or Cdc42/Rho4 can fulfill its role in these events.

Secretory Vesicles Deliver Cdc42p to Sites of Polarized Growth in S. cerevisiae

“…As observed with the anti-Cdc42p antibody, buds and mother cells showed a significant difference (p<0.002; Student’s t-test) in gold labeling, with an average of 31.1 gold particles/um 2 in buds versus 16.3 gold particles/um 2 in mother cells ( Figure 1F ). Although previous studies established the association of Rho1p with secretory vesicles in yeasts [16] – [18] , [29] , these results show the first immunogold localization of Rho1p to 80–100 nm vesicles in any yeast, including S. cerevisiae . Taken together, these data indicate that the immunogold labeling in our studies is antigen-specific and antigen-dependent.…”

Peroxicretion, a Novel Tool for Engineering Membrane Trafficking

“…Peroxisomes are organelles with a single membrane 59 and a very elegant publication shows convincing evidence that peroxisomes originate at the ER 60 . In addition, the identification of the small GTPase Rho1p as being localized on peroxisomes by interaction of the peroxisomal membrane protein Pex25p again indicates a link of peroxisomes with the secretory machinery 61 , since Rho1p is known to play a role in actin reorganization and membrane dynamics 62,63 . In yeast, polarized growth is regulated by Rho1p 64 , and also in fungal systems the link between Rho1 and polarized growth has been confirmed 65 in the filamentous fungus A. niger, polarized growth is linked to secretion of proteins 66 .…”