Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's disease (PD). However, its physiological and pathological functions are unknown. Therefore, we generated several independent Drosophila lines carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes.

Rab GTPases have been implicated in intracellular vesicle trafficking. Using the yeast two-hybrid screen, we have isolated a rat clone that interacts with Rab3A as well as with Rab1. The gene encodes a 20.6-kDa protein with two extensive hydrophobic domains and is broadly expressed in all tissues. This protein binds to prenylated Rab GTPases but not to other small Ras-like GTPases such as the Rho/Rac family. This prenylated Rab acceptor (PRA1) also binds specifically to the synaptic vesicle protein VAMP2 (or synaptobrevin II) but shows no affinity for VAMP1 or cellubrevin in both the yeast twohybrid system and in vitro binding assays. This specificity resides, in part, in the proline-rich domain of VAMP2 as a chimera containing this domain of VAMP2 fused to VAMP1 is able to bind to PRA1. The transmembrane domain of VAMP2 is also essential as its deletion abolished binding to PRA1. Replacement of the deleted VAMP2 transmembrane domain by a CAAX prenylation signal can not restore binding to PRA1. This interaction is therefore distinct from that required for VAMP2 binding to either syntaxin or both syntaxin and SNAP-25. Deletion analysis on PRA1 indicates that the critical Rab-and VAMP2-interacting residues reside in two regions: the amino-terminal residues 30 -54 and the extreme carboxyl-terminal domain. This dual Rab and VAMP2 binding characteristic suggests that PRA1 may serve to link these two protein families in the control of vesicle docking and fusion.

Rab is a family of small Ras-like GTPases regulating intracellular vesicle transport. We have previously reported that prenylated Rab acceptor or PRA1 interacts with Rab GTPases and vesicle-associated membrane protein (VAMP2). Structural prediction programs suggest that PRA1, with its two extensive hydrophobic domains, is likely to be an integral membrane protein. However, subcellular fractionation and immunocytochemical analyses indicated that PRA1 is localized both in the cytosol and tightly associated with the membrane compartment. The membrane-bound form can be partially extracted with physiological buffer and urea, suggesting that PRA1 is an extrinsic membrane protein.Deletion of the carboxyl-terminal domain resulted in a protein that behaved as an integral membrane protein, indicating that this domain plays an essential role in maintaining PRA1 in a soluble state. PRA1 can also bind weakly to GDP dissociation inhibitor (GDI), a protein involved in the solubilization of membrane-bound Rab GTPases. Addition of PRA1 inhibited the extraction of membrane-bound Rab3A by GDI, suggesting that membrane localization of Rab GTPases is dependent on the opposing action of PRA1 and GDI. The binding of Rab and VAMP2 to PRA1 is mutually exclusive such that Rab3A can displace VAMP2 in a preformed VAMP2-PRA1 complex.

The prenylated Rab acceptor (PRA) 1 is a protein that binds prenylated Rab GTPases and inhibits their removal from the membrane by GDI. We describe here the isolation of a second isoform that can also bind Rab GTPases in a guanine nucleotide-independent manner. The two PRA isoforms showed distinct intracellular localization with PRA1 localized primarily to the Golgi complex and PRA2 to the endoplasmic reticulum (ER) compartment. The localization signal was mapped to the COOH-terminal domain of the two proteins. A DXEE motif served to target PRA1 to the Golgi. Mutation of any one of the acidic residues within this motif resulted in significant retention of PRA1 in the ER compartment. Moreover, the introduction of a di-acidic motif to the COOH-terminal domain of PRA2 resulted in partial localization to the Golgi complex. The domain responsible for ER localization of PRA2 was also confined to the carboxyl terminus. Our results showed that these sorting signals were primarily responsible for the differential localization of the two PRA isoforms.

Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (3-6%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be efficiently transported from the ER upon cholesterol depletion. Fluorescence photobleaching recovery experiments indicated that cholesterol depletion by statins leads to a severe loss of lateral mobility on the ER membrane of these transmembrane proteins, but not loss of mobility of proteins in the ER lumen. This impaired lateral mobility is correlated with impaired ER-to-Golgi transport. These results provide evidence for the first time that cholesterol is required in the ER membrane to maintain mobility of membrane proteins and thus protein secretion.