Search PubMed⌕ Search

Biomedical subjects

W E Balch

Publications and source records attributed to W E Balch.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of Sar1-GDP at 1.7 A resolution and the role of the NH2 terminus in ER export.

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the ER. We report the 1.7-A structure of Sar1 and find that consistent with the sequence divergence of Sar1 from Arf family GTPases, Sar1 is structurally distinct. In particular, we show that the Sar1 NH2 terminus contains two regions: an NH2-terminal extension containing an evolutionary conserved hydrophobic motif that facilitates membrane recruitment and activation by the mammalian Sec12 guanine nucleotide exchange factor, and an alpha1' amphipathic helix that contributes to interaction with the Sec23/24 complex that is responsible for cargo selection during ER export. We propose that the hydrophobic Sar1 NH2-terminal activation/recruitment motif, in conjunction with the alpha1' helix, mediates the initial steps in COPII coat assembly for export from the ER.

Animals↗
PubMed2001

Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca(2+) stabilization.

Hereditary familial amyloidosis of Finnish type (FAF) leading to amyloid in the peripheral and central nervous systems stems from deposition of a 71 residue fragment generated from the D187N/Y variants of plasma gelsolin by two sequential endoproteolytic events. We identify the protease accomplishing the first cleavage as furin, a proprotein convertase. Endoproteolysis of plasma gelsolin occurs in the trans-Golgi network due to the inability of the FAF variants to bind and be stabilized by Ca(2+). Secretion and processing of the FAF variants by furin can be uncoupled by blocking the convergence of the exocytic pathway transporting plasma gelsolin and the endocytic recycling of furin. We propose that coincidence of membrane trafficking pathways contributes to the development of proteolysis-initiated amyloid disease.

Amyloidosis, Familial↗
PubMed2001

The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly.

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

Animals↗
PubMed2001

Purification and properties of rat liver Sec23-Sec24 complex.

We have demonstrated a protocol for purifying functional Sec23-24 complex from rat liver cytosol. Because the rat liver Sec23-24 complex is highly susceptible to proteolysis, we have noted several modifications which have allowed us to overcome the problem of degradation. If care is taken to prevent proteolysis, this procedure typically yields 2 mg of functional complex. The Sec23-24 complex can then be used to study Sar1 GTP hydrolysis in the Sec23 GTPase activation assay. Additionally, Sec23-24 can reconstitute ER vesicle formation in the presence of Sar1 and Sec13-31, allowing for the identification of novel proteins or compounds that affect cargo export.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

The mammalian guanine nucleotide exchange factor mSec12 is essential for activation of the Sar1 GTPase directing endoplasmic reticulum export.

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the endoplasmic reticulum of mammalian cells. To begin to elucidate its mechanism of action, we now report the identity of the mammalian homolog to the yeast Sec12 guanine nucleotide exchange factor (18% identity) that promotes Sar1 activation. Mammalian Sec12 (mSec12) is a type II transmembrane protein with a large cytosolic domain, a fragment of which has previously been reported as the transcription factor prolactin regulatory element binding protein (PREB). mSec12 promotes efficient guanine nucleotide exchange on Sar1, but not Arf1 or Rab GTPases. mSec12 is localized to the endoplasmic reticulum and an antibody to the cytosolic domain of mSec12 potently inhibits Sar1 recruitment and the formation of COPII vesicles in vitro. The dominant negative GDP-restricted mutant Sar1[T39N] is shown to be a potent inhibitor of mSec12 activity, consistent with its role in preventing COPII vesicle formation in vitro and during transient expression in vivo. We propose that mSec12 is an evolutionarily distant guanine nucleotide exchange factor directing Sar1 GTPase activation in mammalian cells. Its divergence from yeast Sec12p may reflect the specialized needs of the mammalian endoplasmic reticulum involving the formation of Sar1-dependent transitional elements (Aridor M, et al. J Cell Biol 2001;152:213-229) and selection of cargo into prebudding complexes.

ADP-Ribosylation Factor 1↗
PubMed2001

Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease.

Choroideremia is an X-chromosome-linked disease that leads to the degeneration of the choriocapillaris, the retinal pigment epithelium and the photoreceptor layer in the eye. The gene product defective in choroideremia, CHM, is identical to Rab escort protein 1 (REP1). CHM/REP1 is an essential component of the catalytic geranylgeranyltransferase II complex (GGTrII) that delivers newly synthesized small GTPases belonging to the RAB gene family to the catalytic complex for post-translational modification. CHM/REP family members are evolutionarily related to members of the guanine nucleotide dissociation inhibitor (GDI) family, proteins involved in the recycling of Rab proteins required for vesicular membrane trafficking through the exocytic and endocytic pathways, forming the GDI/CHM superfamily. Biochemical and structural analyses have now revealed a striking parallel in the organization and function of these two families allowing us to generate a general model for GDI/CHM superfamily function in health and disease.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Kinase signaling initiates coat complex II (COPII) recruitment and export from the mammalian endoplasmic reticulum.

The events regulating coat complex II (COPII) vesicle formation involved in the export of cargo from the endoplasmic reticulum (ER) are unknown. COPII recruitment to membranes is initiated by the activation of the small GTPase Sar1. We have utilized purified COPII components in both membrane recruitment and cargo export assays to analyze the possible role of kinase regulation in ER export. We now demonstrate that Sar1 recruitment to membranes requires ATP. We find that the serine/threonine kinase inhibitor H89 abolishes membrane recruitment of Sar1, thereby preventing COPII polymerization by interfering with the recruitment of the cytosolic Sec23/24 COPII coat complex. Inhibition of COPII recruitment prevents export of cargo from the ER. These results demonstrate that ER export and initiation of COPII vesicle formation in mammalian cells is under kinase regulation.

Adenosine Triphosphate↗
PubMed2000

Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion.

The guanosine triphosphatase Rab1 regulates the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus through interaction with effector molecules, but the molecular mechanisms by which this occurs are unknown. Here, the tethering factor p115 was shown to be a Rab1 effector that binds directly to activated Rab1. Rab1 recruited p115 to coat protein complex II (COPII) vesicles during budding from the endoplasmic reticulum, where it interacted with a select set of COPII vesicle-associated SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) to form a cis-SNARE complex that promotes targeting to the Golgi apparatus. We propose that Rab1-regulated assembly of functional effector-SNARE complexes defines a conserved molecular mechanism to coordinate recognition between subcellular compartments.

Animals↗
PubMed2000

Molecular basis for Rab prenylation.

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Delta null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Delta null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6(ts)) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Stage-specific assays to study biosynthetic cargo selection and role of SNAREs in export from the endoplasmic reticulum and delivery to the Golgi.

To analyze the role of coat protein type II (COPII) coat components and targeting and fusion factors in selective export from the endoplasmic reticulum (ER) and transport to the Golgi, we have developed three novel, stage-specific assays. Cargo selection can be measured using a "stage 1 cargo capture assay," in which ER microsomes are incubated in the presence of glutathione S-transferase (GST)-tagged Sar1 GTPase and purified Sec23/24 components to follow recruitment of biosynthetic cargo to prebudding complexes. This cargo recruitment assay can be followed by two sequential assays that measure separately the budding of COPII-coated vesicles from ER microsomes (stage 2) and, finally, delivery of cargo-containing vesicles to the Golgi (stage 3). We show how these assays provide a means to identify the snap receptor (SNARE) protein rBet1 as an essential component that is not required for vesicle formation, but is required for vesicle targeting and fusion during ER-to-Golgi transport. In general, these assays provide an approach to characterize the biochemical basis for the recruitment of a wide variety of biosynthetic cargo proteins to COPII vesicles and the role of different transport components in the early secretory pathway of mammalian cells.

Biological Transport↗
PubMed2000

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 alpha-GDI at ultra-high resolution (1.04 A). Refinement at this resolution highlighted a region with high mobility of its main-chain residues. This corresponded to a surface loop in the primarily alpha-helical domain II at the base of alpha-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 alpha-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.

Amino Acid Motifs↗
PubMed2000

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). delta F508 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 delta F508. 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.

Animals↗