Bryan Moyer

Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering.

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis‐SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis‐Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1‐GTP in a p115‐independent manner and is required for coat protein II vesicle targeting/fusion with the cis‐Golgi. We propose a ‘homing hypothesis’ in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.

COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code

Cystic fibrosis (CF) is a childhood hereditary disease in which the most common mutant form of the CF transmembrane conductance regulator (CFTR) ΔF508 fails to exit the endoplasmic reticulum (ER). Export of wild-type CFTR from the ER requires the coat complex II (COPII) machinery, as it is sensitive to Sar1 mutants that disrupt normal coat assembly and disassembly. In contrast, COPII is not used to deliver CFTR to ER-associated degradation. We find that exit of wild-type CFTR from the ER is blocked by mutation of a consensus di-acidic ER exit motif present in the first nucleotide binding domain. Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24. We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in ΔF508-expressing patients.

Non-conventional Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator through the Early Secretory Pathway

The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-ΔF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000)Traffic 1, 852–870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNApREceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells.

Endoplasmic reticulum retention, degradation, and aggregation of olfactory G-protein coupled receptors.

The mammalian olfactory G‐protein coupled receptor family is comprised of hundreds of proteins that mediate odorant binding and initiate signal transduction cascades leading to the sensation of smell. However, efforts to functionally express olfactory receptors and identify specific odorant ligand–olfactory receptor interactions have been severely impeded by poor olfactory receptor surface expression in heterologous systems. Therefore, experiments were performed to elucidate the cellular mechanism(s) responsible for inefficient olfactory receptor cell surface expression. We determined that the mouse odorant receptors mI7 and mOREG are not selected for export from the ER and therefore are not detectable at the Golgi apparatus or plasma membrane. Specifically, olfactory receptors interact with the ER chaperone calnexin, are excluded from ER export sites, do not accumulate in ER–Golgi transport intermediates at 15u2003°C, and contain endoglycosidase H‐sensitive oligosaccharides, consistent with olfactory receptor exclusion from post‐ER compartments. A labile pool of ER‐retained olfactory receptors are post‐translationally modified by polyubiquitination and targeted for degradation by the proteasome. In addition, olfactory receptors are sequestered into ER aggregates that are degraded by autophagy. Collectively, these data demonstrate that poor surface expression of olfactory receptors in heterologous cells is attributable to a combination of ER retention due to inefficient folding and poor coupling to ER export machinery, aggregation, and degradation via both proteasomal and autophagic pathwaysPlasmids .

Traffic Pattern of Cystic Fibrosis Transmembrane Regulator through the Early Exocytic Pathway

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre‐Golgi intermediates and Golgi compartments, wild‐type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild‐type CFTR is present in pre‐Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2‐fold lower than that found in the endoplasmic reticulum (ER). ΔF508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre‐Golgi intermediates and peripheral cis Golgi elements, but not in post‐Golgi compartments. Our results suggest that the low level of wild‐type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in ΔF508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.

Endoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression

BackgroundResearch on olfactory G-protein coupled receptors (GPCRs) has been severely impeded by poor functional expression in heterologous systems. Previously, we demonstrated that inefficient olfactory receptor (OR) expression at the plasma membrane is attributable, in part, to degradation of endoplasmic reticulum (ER)-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy. Thus, experiments were performed to test the hypothesis that attenuation of ER degradation improves OR functional expression in heterologous cells.ResultsTo develop means to increase the functional expression of ORs, we devised an approach to measure activation of the mOREG OR (Unigene # Mm.196680; Olfr73) through coupling to an olfactory cyclic nucleotide-gated cation channel (CNG). This system, which utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons, was used to demonstrate that degradation, both by the ubiquitin-proteasome system and autophagy, limits mOREG functional expression. The stimulatory effects of proteasome and autophagy inhibitors on mOREG function required export from the ER and trafficking through the biosynthetic pathway.ConclusionsThese findings demonstrate that poor functional expression of mOREG in heterologous cells is improved by blocking proteolysis. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination.

A new functional domain of guanine nucleotide dissociation inhibitor (alpha-GDI) involved in Rab recycling.

Guanine nucleotide dissociation inhibitor (GDI) is a 55‐kDa protein that functions in vesicular membrane transport to recycle Rab GTPases. We have now determined the crystal structure of bovine α‐GDI at ultra‐high resolution (1.04 Å). Refinement at this resolution highlighted a region with high mobility of its main‐chain residues. This corresponded to a surface loop in the primarily α‐helical domain II at the base of α‐GDI containing the previously uncharacterized sequence‐conserved region (SCR) 3A. Site‐directed mutagenesis showed that this mobile loop plays a crucial role in binding of GDI to membranes and extraction of membrane‐bound Rab. This domain, referred to as the mobile effector loop, in combination with Rab‐binding residues found in the multi‐sheet domain I at the apex of α‐GDI may provide flexibility for recycling of diverse Rab GTPases. We propose that conserved residues in domains I and II synergize to form the functional face of GDI, and that domain II mediates a critical step in Rab recycling during vesicle fusion.

Structural and functional analysis of the globular head domain of p115 provides insight into membrane tethering.

Molecular tethers have a central role in the organization of the complex membrane architecture of eukaryotic cells. p115 is a ubiquitous, essential tether involved in vesicle transport and the structural organization of the exocytic pathway. We describe two crystal structures of the N-terminal domain of p115 at 2.0 A resolution. The p115 structures show a novel alpha-solenoid architecture constructed of 12 armadillo-like, tether-repeat, alpha-helical tripod motifs. We find that the H1 TR binds the Rab1 GTPase involved in endoplasmic reticulum to Golgi transport. Mutation of the H1 motif results in the dominant negative inhibition of endoplasmic reticulum to Golgi trafficking. We propose that the H1 helical tripod contributes to the assembly of Rab-dependent complexes responsible for the tether and SNARE-dependent fusion of membranes.

A new frontier in pharmacology: the endoplasmic reticulum as a regulated export pathway in health and disease

The endoplasmic reticulum (ER), the first secretory compartment of eukaryotic cells, co-ordinates the biogenesis and export of all membrane-bound and soluble cargo molecules to the cell surface. ER function is now recognised to have unprecedented links with signalling pathways regulating cell growth and differentiation and host physiology. Misfolding and aggregation of newly synthesised proteins in the ER or alterations in ER processing of cargo mediated by pathogens is responsible for a broad range of diseases including cystic fibrosis, emphysema and neuropathies such as Alzheimer’s disease. The central, integrative role of the ER in determining cell physiology in health and disease represents an untapped area for pharmacological intervention. This review focuses on the potential use of pharmacological agents to modulate cargo selection, folding and degradation in the ER with the goal of alleviating ER export disease. In addition, implementation of novel technologies that utilise normal ER function to store and release biologically active substances of therapeutic relevance are presented as a new frontier in drug delivery.

1 - Structural Basis for Rab Function: An Overview

Rab proteins, members of the Ras superfamily of low molecular weight guanosine-5-triphosphate (GTP)-binding proteins (20–25 kDa), modulate tubulovesicular trafficking between compartments of the biosynthetic and endocytic pathways. Similar to Ras, Rab GTPases cycle between active GTP-bound and inactive guanosine diphosphate GDP-bound states. This chapter describes the Rab structure–function relationships in the context of membrane trafficking and serves as a prelude for the accompanying chapters, which describe specific methods for elucidating Rab function. The unifying theme in research elucidating Rab structure-function relationships has been the Rab GTPase cycle model. In the cytosol, Rab proteins are maintained in the GDP-bound state by interaction with a GDP dissociation inhibitor (GDI). GDI delivers Rab-GDP to donor membranes where GDI may be displaced by a GDI displacement factor (GDF). Subsequently, a guanine nucleotide exchange factor (GEF) is believed to stimulate exchange of GDP for GTP. Transport intermediates containing activated Rab bud from donor membranes, where Rab-GTP recruits effector molecules required for trafficking to acceptor compartments. Recent studies suggest that Rab effectors regulate the motility of transport intermediates along cytoskeletal elements and mediate the docking/fusion of transport intermediates with acceptor membranes.