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

Abstract

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.

Presynaptic precursor vesicles originate from the trans-Golgi network, promoted by the small GTPase RAB2

“…RAB2 is described as a Golgi resident and its depletion in some cell types reported to affect Golgi apparatus integrity (Aizawa and Fukuda, 2015;Maringer et al, 2016). However, when comparing Golgi marker intensity and Golgi number of rab2 mutants and wild type, no significant differences were detectable ( Fig.…”

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

“…DdRabS has been shown to localize between the ER-Golgi membranes and the CV system [ 15 ], and it also partially colocalizes with endosomes. DdRabS(DN) cells colocalize to endosomes and lysosomes, and it has been hypothesized that DdRabS may also be involved in trafficking along the endo-lysosomal pathway as well.…”

“…The DdRabS(DN) cell line was created using the Stratagene QuickChange ® II Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA). The resulting plasmid was also transformed into AX4 and selected for with G418 [ 15 ].…”

“…It was previously observed that DdRabS localizes to ER-Golgi membranes and the contractile vacuole system [ 15 ]. This protein is a Rab GTPase that is somewhat homologous to Rab1 in other organisms; as is commonly done in this organism, proteins with a lower degree of homology are typically given a letter as a name while those with higher homology use a number and additional letters for any existing isoforms.…”

Evolution of protein trafficking in kinetoplastid parasites: Complexity and pathogenesis

“…In fact, these two types of organelles are often associated with each other physically and functionally, and while maintaining their membrane individual identities, are both subject to regulation by Rab8 (absent in all kinetoplastids) and the exocyst complex . Additionally, there are other lines of evidence that suggest selective trafficking of proteins between CVs and the plasma membrane; several trafficking and vacuolar fusion proteins were also observed in the T. cruzi CV including SNARE VAMP7A, Rab11, Rab2 and Rab1 …”