Thirty-One Flavors of Drosophila Rab Proteins

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ABSTRACT

Rab proteins are small GTPases that play important roles in transport of vesicle cargo and recruitment, association of motor and other proteins with vesicles, and docking and fusion of vesicles at defined locations. In vertebrates, >75 Rab genes have been identified, some of which have been intensively studied for their roles in endosome and synaptic vesicle trafficking. Recent studies of the functions of certain Rab proteins have revealed specific roles in mediating developmental signal transduction. We have begun a systematic genetic study of the 33 Rab genes in Drosophila. Most of the fly proteins are clearly related to specific vertebrate proteins. We report here the creation of a set of transgenic fly lines that allow spatially and temporally regulated expression of Drosophila Rab proteins. We generated fluorescent protein-tagged wild-type, dominant-negative, and constitutively active forms of 31 Drosophila Rab proteins. We describe Drosophila Rab expression patterns during embryogenesis, the subcellular localization of some Rab proteins, and comparisons of the localization of wild-type, dominant-negative, and constitutively active forms of selected Rab proteins. The high evolutionary conservation and low redundancy of Drosophila Rab proteins make these transgenic lines a useful tool kit for investigating Rab functions in vivo.

THE process of intracellular transport is important for almost every aspect of cellular function and for proper organism development. In highly compartmentalized eukaryotic cells, a large group of monomeric small GTPases, termed Rab proteins, orchestrate vesicle trafficking among distinct cellular membrane compartments, including cargo selection, vesicle budding, moving, tethering, docking, and targeting (Pfeffer 2001; Pfeffer and Aivazian 2004; Ali and Seabra 2005; Jordens et al. 2005; Pfeffer 2005). Rab proteins are members of the larger family of Ras-like GTPases, which regulate vesicle trafficking, transmembrane signal transduction, and cytoskeletal rearrangements, among other functions (Satoh et al. 1992a,b; Hernandez-Alcoceba et al. 2000).

Like most other small GTPases, Rab proteins undergo two alternate conformational transitions upon binding to either GDP or GTP. In response to signal stimuli, guanine nucleotide exchange factors interact with Rab GTPases, trigger their binding to GTP, and enable their interactions with various targets and effector proteins. GTPase-activating proteins work in the opposite direction, accelerating GTP hydrolysis and leaving GDPbound Rab proteins inactive. In the GTP-bound active form, each Rab can interact with a different complex of proteins (effectors) to facilitate the delivery of transport vesicles to different acceptor membranes (Molendijk et al. 2004; Pfeffer and Aivazian 2004).

Mutations in Rab genes can affect cell growth, motility, and other biological processes. The first member of the Rab subfamily GTPases to be studied, Sec4p, was identified in yeast as an essential protein required for secretory vesicle exocytosis (Salminen and Novick 1987). Mammalian relatives of this yeast protein were identified and formally designated Rab (ras-like genes in rat brain) proteins. Different Rab proteins are found to be specifically associated with distinct subcellular membrane compartments and some have become standard markers for these compartments. Rab1 is present in the endoplasmic reticulum, Rab6 in the Golgi, Rab3 in synaptic vesicles, Rab5 in early endosomes, Rab7 and Rab9 in late endosomes, and Rab11 in the recycling endosome (Pfeffer 2001; Pfeffer and Aivazian 2004; Ali and Seabra 2005; Jordens et al. 2005; Pfeffer 2005).

Mutations affecting Rab GTPases and their regulatory proteins and effectors have been identified in multiple developmental disorders and malignancies. These include Griscelli syndrome, an autosomal recessive disorder caused by a mutation in Rab27a and characterized by pigment dilution in the hair and uncontrolled T-cell activation; choroideremia, an X-linked form of retinal degeneration with slow onset and progression caused by a mutation in Rab escort protein-1; and Hermansky- Pudlak syndrome, an autosomal recessive disorder caused by a mutation in Rab geranylgeranyl transferase and characterized by partial albinism and a tendency to bleed (Pereira-Leal et al. …