Disruption of Golgi Morphology and Trafficking in Cells Expressing Mutant Prenylated Rab Acceptor-1

Abstract

Prenylated Rab acceptor (PRA1) is a protein that binds Rab GTPases and the v-SNARE VAMP2. The protein is localized to the Golgi complex and post-Golgi vesicles. To determine its functional role, we generated a number of point mutations and divided them into three classes based on cellular localization. Class A mutants were retained in the endoplasmic reticulum (ER) and exerted an inhibitory effect on transport of vesicular stomatitis virus envelope glycoprotein (VSVG) from the ER to Golgi as well as to the plasma membrane. Class B mutants exhibited a highly condensed Golgi complex and inhibited exit of anterograde cargo from this organelle. Class C mutants exhibited an intermediate phenotype with Golgi and ER localization along with extensive tubular structures emanating from the Golgi complex. There was a direct correlation between the cellular phenotype and binding to Rab and VAMP2. Class A and C mutants showed a significant decrease in Rab and VAMP2 binding, whereas an increase in binding was observed in the class B mutants. Thus, PRA1 is required for vesicle formation from the Golgi complex and might be involved in recruitment of Rab effectors and SNARE proteins during cargo sequestration.Rab GTPases constitute the largest group within the Ras superfamily. They regulate vesicle trafficking by cycling through active membrane-bound GTP-and inactive cytosolic GDP-bound states. Membrane localization requires modification of the cysteine-containing motif at the carboxyl terminus by prenyl residues. Cycling between the membrane and cytosol is mediated by GDP dissociation inhibitor (GDI), 1 which extracts GDP-bound Rab from the membrane. Activation through guanine nucleotide exchange at the membrane is catalyzed by a guanine nucleotide exchange factor, of which a number have been identified in mammals (1, 2).Vesicular transport through the secretory pathway undergoes a number of discrete steps each involving budding, membrane remodeling, targeting, docking, and fusion. In ER to Golgi transport, anterograde cargo proteins such as VSVG are selectively transported to the Golgi along with resident ER proteins with the latter retrieved by a salvage process that recognizes distinct motifs within the protein (3). These transport vesicles contain an electron-dense coat assembled under the control of the small GTPase ARF (4). The fungal metabolite brefeldin A (BFA) inhibits ARF activation by stabilizing the inactive ARF-guanine nucleotide exchange factor (GEF) complex (5) resulting in retrograde transport of Golgi content to the ER. At the Golgi complex, cargo proteins destined for postGolgi locations are sorted into distinct carriers upon exit from the trans face, whereas Golgi resident proteins such as mannosidase II (Man II) are selectively retrieved in COPI-coated vesicles and returned to the cis face (6).Vesicle fusion is mediated by the core SNARE complex consisting of the vesicle protein VAMP, or synaptobrevin, and two other membrane proteins, syntaxin and SNAP-25 (7). Rab effectors play a regulatory role in this pro...

A role for prenylated rab acceptor 1 in vertebrate photoreceptor development

“…The association between vesicular trafficking defects and retinal degenerative disease is consistent with the early pathology observed in the rd1 mouse. PRA1 has been proposed to regulate the recruitment of Rab effector proteins as well as proteins involved in proper tethering and fusion of vesicles to their target membrane, such as VAMP2 [12,55]. A defect in recruitment by PRA1 of Rab effectors and proteins involved in the downstream events of vesicular trafficking could correlate to the defects in vesicular trafficking reported in rd1 retinas [17,56].…”

JM4 is a four‐transmembrane protein binding to the CCR5 receptor

“…This family of proteins includes also GTRAP3-18. PRA1 regulates vesicle biogenesis at the Golgi [35] and like ARFs associates with membranous vesicles during assembly and transport [36]. Furthermore, PRA1 has just recently been shown to interact with a GPCR [37], underscoring its relationship with JM4.…”

Efficacy of MS-275, a selective inhibitor of class I histone deacetylases, in human colon cancer models

“…These markers were up-regulated dose-dependently upon MS-275 treatment in the five responder models and showed no induction in the three non-responder models. PRA1 is a ubiquitous protein which binds to prenylated Rab GTPases (42). Its overexpression impairs TCF/ß-catenin signaling, which plays a prominent role in colon cancer, possibly by limiting nuclear translocation of ß-catenin (43).…”