The Effect of Overexpressed DdRabS on Development, Cell Death, Vesicular Trafficking, and the Secretion of Lysosomal Glycosidase Enzymes.

Rab GTPases are essential regulators of many cellular processes and play an important role in downstream signaling vital to proper cell function. We sought to elucidate the role of novel D. discoideum GTPase RabS. Cell lines over-expressing DdRabS and expressing DdRabS N137I (dominant negative (DN)) proteins were generated, and it was determined that DdRabS localized to endosomes, ER-Golgi membranes, and the contractile vacuole system. It appeared to function in vesicular trafficking, and the secretion of lysosomal enzymes. Interestingly, microscopic analysis of GFP-tagged DdRabS (DN) cells showed differential localization to lysosomes and endosomes compared to GFP-tagged DdRabS overexpressing cells. Both cell lines over-secreted lysosomal glycosidase enzymes, especially ß-glucosidase. Furthermore, DdRabS overexpressing cells were defective in aggregation due to decreased cell⁻cell cohesion and sensitivity to cAMP, leading to abnormal chemotactic migration, the inability to complete development, and increased induced cell death. These data support a role for DdRabS in trafficking along the vesicular and biosynthetic pathways. We hypothesize that overexpression of DdRabS may interfere with GTP activation of related proteins essential for normal development resulting in a cascade of defects throughout these processes.

Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation.

Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium discoideum. DdRabS is a Dictyostelium Rab that is 80 percent homologous to DdRab1 which is required for protein transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms, affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system in Dictyostelium.

Osh6 overexpression extends the lifespan of yeast by increasing vacuole fusion.

In yeast cells, the vacuole divides and fuses in each round of cell cycle. While mutants defective in vacuole fusion are "wild type" for vegetative growth, most have shortened replicative lifespans under caloric restriction (CR) condition, a manipulation that extends lifespan in wild type cells. To explore whether vacuole fusion extends lifespan, we screened for genes that can complement the fusion defect of selected mutants (erg6Δ, a sterol mutant; nyv1Δ,  a mutant involved in the vacuolar SNARE complex and vac8Δ, a vacuolar membrane protein mutant). This screen revealed that Osh6, a member of the oxysterol-binding protein family, can complement the vacuole fusion defect of nyv1Δ, but not erg6Δ or vac8Δ, suggesting that Osh6's function in vacuole fusion is partly dependent on membrane ergosterol and Vac8. To measure the effect of OSH6 on lifespan, we replaced the endogenous promoter of OSH6 with a shorter version of the ERG6 promoter to obtain PERG6-OSH6. This mutant construct significantly extended the replicative lifespan in a wild type background and in a nyv1Δ mutant. Interestingly, PERG6-OSH6 cells were more sensitive to drugs that inhibit the activity of the TOR complex 1 (TORC1) than wild type cells. Moreover, a PERG6-OSH6 tor1Δ double mutant demonstrated a greatly shortened lifespan, suggesting a genetic interaction between Osh6 and Tor1. Since active TORC1 stimulates vacuole scission and CR downregulates TORC1, Osh6 may link these two pathways by adjusting vacuolar membrane organization to extend lifespan.