Biomedical subjects

J M McCaffery

Publications and source records attributed to J M McCaffery.

At least 37 records · Page 2Linked to original sources

PubMed1999

Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.

Amino Acid Sequence↗

PubMed1999

Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro.

Galpha-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for Galpha subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIP-enriched vesicles could accelerate the GTPase activity of Galphai proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5x) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant Galphai3 and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant Galphai3 protein increased the GTPase activity. We conclude that CCVs possess GAP activity for Galphai3 and that membrane-associated GAIP is capable of interacting with Galphai3. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.

Animals↗

PubMed1999

A signal recognition particle receptor gene from the early-diverging eukaryote, Giardia lamblia.

The molecular mechanisms for targeting and translocation of secreted proteins are highly conserved from bacteria to mammalian cells, although the machinery is more complex in higher eukaryotes. To investigate protein transport in the early-diverging eukaryote, Giardia lamblia, we cloned the gene encoding the alpha subunit (SRalpha) of the signal recognition particle (SRP) receptor. SRalpha is a small GTPase that functions in SRP-ribosome targeting to the ER. Sequence and phylogenetic analyses showed that SRalpha from G. lamblia is most homologous to SRalpha proteins from higher eukaryotes, although it lacks some conserved motifs. Specifically, giardial SRalpha has an N-terminal extension that enables SRalpha of higher eukaryotes to interact with a beta subunit that anchors it in the ER membrane. While the C-terminal regions are similar, giardial SRalpha lacks a prominent 13 amino acid regulatory loop that is characteristic of higher eukaryotic versions. Thus, giardial SRalpha resembles that of higher eukaryotes, but likely diverged before the advent of the regulatory loop. The 1.8 kb SRalpha transcript has extremely short untranslated regions (UTRs): a 1-2 nt 5'- and a 9 nt 3' UTR with the polyadenylation signal overlapping with the stop codon. RT-PCR, Northern and Western analyses showed that SRalpha is present at relatively constant levels during vegetative growth and encystation, even though there are extensive changes in endomembrane structures and secretory activity during encystation. Imnuno-EM showed that SRalpha localizes to ER-like structures, strengthening the observation of a typical ER in G. lamlia. Unexpectedly, SRalpha was also found in the lysosome-like peripheral vacuoles, suggesting unusual protein traffic in this early eukaryote. Our results indicate that the eukaryotic type of cotranslational transport appeared early in the evolution of the eukaryotic cell.

Amino Acid Sequence↗

PubMed1999

The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast.

The dynamin-related GTPase Dnm1 controls mitochondrial morphology in yeast. Here we show that dnm1 mutations convert the mitochondrial compartment into a planar 'net' of interconnected tubules. We propose that this net morphology results from a defect in mitochondrial fission. Immunogold labelling localizes Dnm1 to the cytoplasmic face of constricted mitochondrial tubules that appear to be dividing and to the ends of mitochondrial tubules that appear to have recently completed division. The activity of Dnm1 is epistatic to that of Fzo1, a GTPase in the outer mitochondrial membrane that regulates mitochondrial fusion. dnm1 mutations prevent mitochondrial fragmentation in fzo1 mutant strains. These findings indicate that Dnm1 regulates mitochondrial fission, assembling on the cytoplasmic face of mitochondrial tubules at sites at which division will occur.

Crosses, Genetic↗

PubMed1999

Genetic and environmental causes of covariation among blood pressure, body mass and serum lipids during young adulthood: a twin study.

OBJECTIVE: To determine the extent to which the correlation of systolic blood pressure, diastolic blood pressure, body mass, fasting total cholesterol and fasting triglycerides in young adulthood reflects common genetic or environmental influences. DESIGN: Cardiovascular risk factors were measured in a community sample of 129 monozygotic and 67 dizygotic twin pairs, ages 18-30 years. METHODS: Multivariate twin structural equation modelling allows estimation of the extent to which the covariation of two or more variables is attributable to common genetic and environmental factors and was used to analyse the correlation among systolic blood pressure, diastolic blood pressure, body mass index, fasting total cholesterol and triglycerides. RESULTS: The covariation of risk factors was partially attributable to a single common genetic factor, while the covariation of systolic blood pressure, body mass index and triglycerides was also, in part, attributable to a common non-shared environmental factor. CONCLUSIONS: Genetic and, to a lesser extent, non-shared environmental factors contribute to the covariation of cardiovascular risk factors in young adult twins. Nonetheless, it should be noted that these common influences account for a relatively small percentage of the variance in each risk factor compared to genetic and environmental factors that are risk factor-specific.

Adolescent↗

PubMed1999

Intracellular localization and membrane topology of 11-cis retinol dehydrogenase in the retinal pigment epithelium suggest a compartmentalized synthesis of 11-cis retinaldehyde.

11-cis retinol dehydrogenase (EC 1.1.1.105) catalyses the last step in the biosynthetic pathway generating 11-cis retinaldehyde, the common chromophore of all visual pigments in higher animals. The enzyme is abundantly expressed in retinal pigment epithelium of the eye and is a member of the short chain dehydrogenase/reductase superfamily. In this work we demonstrate that a majority of 11-cis retinol dehydrogenase is associated with the smooth ER in retinal pigment epithelial cells and that the enzyme is an integral membrane protein, anchored to membranes by two hydrophobic peptide segments. The catalytic domain of the enzyme is confined to a lumenal compartment and is not present on the cytosolic aspect of membranes. Thus, the subcellular localization and the membrane topology of 11-cis retinol dehydrogenase suggest that generation of 11-cis retinaldehyde is a compartmentalized process.

Alcohol Oxidoreductases↗

PubMed1998

A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast.

We have recently characterized three yeast gene products (Vps35p, Vps29p, and Vps30p) as candidate components of the sorting machinery required for the endosome-to-Golgi retrieval of the vacuolar protein sorting receptor Vps10p (Seaman, M.N.J., E.G. Marcusson, J.-L. Cereghino, and S.D. Emr. 1997. J. Cell Biol. 137:79-92). By genetic and biochemical means we now show that Vps35p and Vps29p interact and form part of a multimeric membrane-associated complex that also contains Vps26p, Vps17p, and Vps5p. This complex, designated here as the retromer complex, assembles from two distinct subcomplexes comprising (a) Vps35p, Vps29p, and Vps26p; and (b) Vps5p and Vps17p. Density gradient fractionation of Golgi/endosomal/vesicular membranes reveals that Vps35p cofractionates with Vps5p/Vps17p in a vesicle-enriched dense membrane fraction. Furthermore, gel filtration analysis indicates that Vps35p and Vps5p are present on a population of vesicles and tubules slightly larger than COPI/coatomer-coated vesicles. We also show by immunogold EM that Vps5p is localized to discrete regions at the rims of the prevacuolar endosome where vesicles appear to be budding. Size fractionation of cytosolic and recombinant Vps5p reveals that Vps5p can self-assemble in vitro, suggesting that Vps5p may provide the mechanical impetus to drive vesicle formation. Based on these findings we propose a model in which Vps35p/Vps29p/Vps26p function to select cargo for retrieval, and Vps5p/Vps17p assemble onto the membrane to promote vesicle formation. Conservation of the yeast retromer complex components in higher eukaryotes suggests an important general role for this complex in endosome-to-Golgi retrieval.

Carrier Proteins↗

PubMed1998

The mammalian calcium-binding protein, nucleobindin (CALNUC), is a Golgi resident protein.

We have identified CALNUC, an EF-hand, Ca2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Galphai3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Galphai3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca2+-binding protein detected by 45Ca2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.

Amino Acid Sequence↗

PubMed1998

Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins.

Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/ V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl- serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

Animals↗

PubMed1998

A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris.

Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Delta mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Delta cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Delta mutant and were impaired in binding to the PTS2 sequence.

Acetyl-CoA C-Acyltransferase↗

PubMed1998

Cellular and transcriptional changes during excystation of Giardia lamblia in vitro.

Excystation of Giardia lamblia entails differentiation of dormant cysts into parasitic trophozoites. Despite its importance for infection, this transformation is not understood at the cellular or molecular levels. In these studies, we report that excystation entails detection of environmental stimuli across the tough extracellular cyst wall leading to highly coordinated physiological, structural, and molecular responses. We found that novel cytoplasmic rearrangements and changes in individual species of mRNA and in cytoplasmic pH occur within the cyst wall in the earliest stage of excystation, in response to conditions modeling cyst ingestion and passage into the human stomach. This suggests that cysts do not contain all the mRNA needed for excystation and emergence and supports our hypothesis that external stimuli, including hydrogen ions, may penetrate or be perceived across the cyst wall. In contrast, changes in cyst wall structure or proteins were detected only later in excystation, in the stage that models passage into the human small intestine, where trophozoites can emerge and survive. These findings show that excystation of G. lamblia is a highly complex and active process and provide important insights into its cellular and molecular components.

Animals↗

PubMed1998

Aggression, impulsivity, and central nervous system serotonergic responsivity in a nonpatient sample.

To test the hypothesis that traits of aggression and impulsivity correlate negatively with central serotonergic system function in a nonpatient population, a standard fenfluramine challenge (for assessment of serotonergic responsivity) and behavioral measurements germane to aggression/impulsivity were administered to a community-derived sample of 119 men and women. In men, peak prolactin responses to fenfluramine correlated significantly with an interview-assessed life history of aggression (r = -.40, p < .002), the Barratt Impulsiveness Scale (r = -.30, p < .03), and traits of Conscientiousness (r = +.30, p < .03), Neuroticism (r = -.31, p < .02) and Angry Hostility (r = -.35, p < .01) on the NEO-Personality Inventory. No significant relationships were observed across all women, although subanalyses restricted to postmenopausal subjects (in whom ovarian influences on prolactin secretion may be mitigated because of diminished estrogen) showed a pattern of behavioral associations somewhat similar to that seen in men. By extending documented relationships between an index of central serotonergic system function and traits of aggression and impulsivity to a more normative range of population variability than is represented in prior literature, this study supports speculation that these associations reflect a basic neurobehavioral dimension of individual differences.

Adult↗

PubMed1998

Differentiation-associated surface antigen variation in the ancient eukaryote Giardia lamblia.

Encystation of Giardia lamblia is required for survival outside the host, whereas excystation initiates infection. The dormant cyst was considered an adaptation to external survival and passage through the stomach. However, we found previously that trophozoites which had recovered after completion of the life cycle had switched their major variant surface protein (VSP), called TSA 417, but neither the timing nor the molecular mechanism of switching had been elucidated. Here we demonstrate that TSA 417 predominates in cysts, but is downregulated during the stage of excystation that models cyst arrival in the small intestine. Transcripts of new VSPs appear late in encystation, and during and after excystation. Trophozoites appear to prepare for switching during encystation, when the major VSP on the cell surface diminishes and is internalized in lysosome-like vacuoles. As short-range DNA rearrangements were not detected, giardial VSP switching during differentiation appears to resemble the in situ switching of surface glycoproteins in African trypanosomes. We also report a unique extended 15 nucleotide polyadenylation signal in all VSP transcripts, but not in other known giardial genes. Antigenic variation during encystation-excystation may be a novel form of immune evasion that could help explain the common occurrence of reinfection by Giardia and other parasites with similar life cycles.

Animals↗

PubMed1998

RGS-GAIP, a GTPase-activating protein for Galphai heterotrimeric G proteins, is located on clathrin-coated vesicles.

RGS-GAIP (Galpha-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Galphai/Galphaq-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Galphai3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.

Animals↗

PubMed1997

Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

Amino Acid Sequence↗

PubMed1997

Behaviorally elicited heart rate reactivity and atherosclerosis in ovariectomized cynomolgus monkeys (Macaca fascicularis).

It has been hypothesized that atherogenesis is accelerated among individuals who exhibit heightened cardiovascular reactions to psychologic stress. We have reported previously that the coronary atherosclerosis of cholesterol-fed, male and reproductively intact (premenopausal) female cynomolgus monkeys was exacerbated in animals that experienced the largest heart rate (HR) reactions to a fear-eliciting laboratory stressor. In this article, we report a similar relationship among 20 female monkeys that were rendered estrogen-deficient (by ovariectomy) and subsequently treated with replacement of both estrogen and progesterone. At the beginning of a 30-month study period, animals were fitted with ECG telemetry devices, and their HRs were recorded under baseline and stressed conditions. Stress HR measurements were obtained during a standard challenge involving threatened capture and physical handling of the animals. As part of a related experiment, monkeys were then ovariectomized and, for the remainder of the study, administered 17 beta-estradiol (continuously) and progesterone (cyclically) by subcutaneous Silastic implant (Dow Corning). Animals consumed a cholesterol-containing diet throughout, and HR measurements were repeated in the 24th month. At necropsy, the magnitude of animals' HR responses to stress correlated significantly with intimal area measurements in the left anterior descending and circumflex coronary arteries (r = .59 and r = .57, respectively; P < .009). This association was due to a marked exacerbation of coronary atherosclerosis in animals comprising the upper third of the reactivity distribution. Although total and HDL cholesterol concentrations also covaried with HR reactivity, the greater atherosclerosis of "high" HR reactors persisted after statistical adjustment for concomitant variability in plasma lipids. HR reactivity was unrelated to blood pressure, body weight, or social behavior.

Animals↗

PubMed1996

Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas.

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN-enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).

ADP-Ribosylation Factors↗

PubMed1996

COPII vesicles derived from mammalian endoplasmic reticulum microsomes recruit COPI.

ER to Golgi transport requires the function of two distinct vesicle coat complexes, termed COPI (coatomer) and COPII, whose assembly is regulated by the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, respectively. To address their individual roles in transport, we have developed a new assay using mammalian microsomes that reconstitute the formation of ER-derived vesicular carriers. Vesicles released from the ER were found to contain the cargo molecule vesicular stomatitis virus glycoprotein (VSV-G) and p58, an endogenous protein that continuously recycles between the ER and pre-Golgi intermediates. Cargo was efficiently sorted from resident ER proteins during vesicle formation in vitro. Export of VSV-G and p58 were found to be exclusively mediated by COPII. Subsequent movement of ER-derived carriers to the Golgi stack was blocked by a trans-dominant ARF1 mutant restricted to the GDP-bound state, which is known to prevent COPI recruitment. To establish the initial site of coatomer assembly after export from the ER, we immunoisolated the vesicular intermediates and tested their ability to recruit COPI. Vesicles bound coatomer in a physiological fashion requiring an ARF1-guanine nucleotide exchange activity. These results suggest that coat exchange is an early event preceding the targeting of ER-derived vesicles to pre-Golgi intermediates.

ADP-Ribosylation Factor 1↗