Search PubMed⌕ Search

Biomedical subjects

J E Rothman

Publications and source records attributed to J E Rothman.

At least 145 records · Page 8Linked to original sources
PubMed1986

Multiple cytosolic components promote intra-Golgi protein transport. Resolution of a protein acting at a late stage, prior to membrane fusion.

Cytosolic components are required to produce the "primed donor" and to consume the "dilution-resistant" intermediates of the intercompartmental protein transport pathway as elucidated in a cell-free system (Balch, W. E., Glick, B. S., and Rothman, J. E. (1984) Cell 39, 525-536, and Wattenberg, B. W., Balch, W. E., and Rothman, J. E. (1986) J. Biol. Chem. 261, 2202-2207). Widely different levels of crude cytosol are required for each of these steps, suggesting that different cytosolic components might mediate each step. Here, we fractionate cytosol and demonstrate that there are multiple transport-active components. Furthermore, we report the development of stage-specific functional assays which reveal that a distinct soluble component is required in the consumption of the dilution-resistant intermediate. This component, of about 25 kilodaltons in its apparent native molecular mass, is derived from calf brain cytosol. While this component mediates the consumption of the dilution-resistant intermediate, it is inactive in the priming stage. This stage-specific component seems likely to be involved in the processing of transport vesicles after the attachment of those vesicles to the target membranes.

Animals↗
PubMed1986

Yeast and mammals utilize similar cytosolic components to drive protein transport through the Golgi complex.

Vesicular transport between successive compartments of the mammalian Golgi apparatus has recently been reconstituted in a cell-free system. In addition to ATP, transport requires both membrane-bound and cytosolic proteins. Here we report that the cytosol fraction from yeast will efficiently substitute for mammalian cytosol. Mammalian cytosol contains several distinct transport factors, which we have distinguished on the basis of gel filtration and ion-exchange chromatography. Yeast cytosol appears to contain the same collection of transport factors. Resolved cytosol factors from yeast and mammals complement each other in a synergistic manner. These findings suggest that the molecular mechanisms of intracellular protein transport have been conserved throughout evolution. Moreover, this hybrid cell-free system will enable the application of yeast genetics to the identification and isolation of cytosolic proteins that sustain intracellular protein transport.

Animals↗
PubMed1985

Enzymatic dissociation of clathrin cages in a two-stage process.

Uncoating ATPase catalyzes the ATP-dependent dissociation of clathrin from coated vesicles and empty cages. Following an uncoating reaction, clathrin triskelions are released intact, in a stoichiometric complex with bound uncoating protein. This overall uncoating process was dissected into two partial reactions. In the first, ATP hydrolysis drives the transient displacement of a portion of a triskelion from a cage. Uncoating protein then captures the displaced triskelion, in the second stage, by binding to a newly exposed site on clathrin that had previously been buried in the cage lattice. Triskelion-uncoating protein complexes are released when all points of attachment of the triskelion to the cage have been severed. The uncoating protein interacts with a distinct site on clathrin for each of these reactions.

Adenosine Triphosphatases↗
PubMed1985

Two classes of binding sites for uncoating protein in clathrin triskelions.

Clathrin released from coated vesicles or empty cages by the ATP-dependent action of uncoating protein exists as a complex with the uncoating protein. Despite its apparent consumption during a round of uncoating, we have found that uncoating protein functions as an enzyme in that it rapidly and spontaneously recycles from its product (triskelions) to its substrate (cages). The binding of uncoating protein to clathrin triskelions is a complex equilibrium that involves the interaction of uncoating protein with at least two distinct sites on the clathrin molecule. Limited proteolysis dissected clathrin into two domains, each of which contained distinct binding sites. Binding to one of these sites, located on the proximal leg of a triskelion, was dependent upon the presence of light chains and was unstable to gel filtration. Binding to the second kind of site, located on the distal portion of a triskelion leg, was stable to gel filtration and was independent of the presence of light chains.

Adenosine Triphosphatases↗
PubMed1985

ATP catalyzes the sequestration of clathrin during enzymatic uncoating.

ATP facilitates the sequestration of displaced triskelions by uncoating protein. In so doing, ATP is not hydrolyzed; nor does the concentration of ATP affect the equilibrium of this binding. However, the rates of both the binding of uncoating protein to clathrin and of their dissociation are greatly accelerated by ATP. These properties suggest that ATP acts catalytically to speed the capture of displaced triskelions by uncoating protein, as well as stoichiometrically in its hydrolysis to drive the displacement of triskelions from cages. The nucleotide specificity of this "catalytic" site for ATP on the uncoating protein is much less strict than that of the distinct "hydrolytic" site that drives the ATP-dependent displacement of triskelions from cages.

Adenosine Triphosphate↗
PubMed1985

Glucosidase II, a glycoprotein of the endoplasmic reticulum membrane. Proteolytic cleavage into enzymatically active fragments.

Glucosidase II removes the inner two alpha-linked glucose residues from freshly transferred Asn-linked oligosaccharide chains in the endoplasmic reticulum. This enzyme, whose activity could be measured by the hydrolysis of an artificial substrate (p-nitrophenyl alpha-D-glucopyranoside), was purified 240-fold from a rat liver microsome fraction by DEAE-cellulose, concanavalin A-Sepharose 4B, and hydroxylapatite chromatography. The apparent molecular weight of the active polypeptide was 123 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Glucosidase II has at least one high-mannose oligosaccharide chain that can be cleaved by endoglycosidase H. Trypsin readily cleaved the 123-kilodalton (kDa) form of glucosidase II into a fully active 73-kDa core. The pattern of this cleavage suggests a domain structure for this enzyme. We demonstrate that trypsin first removes a glycosylated 25-kDa domain to yield an apparently unglycosylated 98-kDa product which is further cleaved to yield the active 73-kDa core.

Animals↗
PubMed1985

Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system.

Transport of the vesicular stomatitis virus (VSV)-encoded glycoprotein (G protein) between successive compartments of the Golgi in a cell-free system is measured by the coupled incorporation of N-[3H]acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported from a "donor" compartment in Golgi membranes that lack GlcNAc transferase I (from VSV-infected CHO clone 15B cells) to the next "acceptor" compartment in a Golgi population from wild-type CHO cells (containing the GlcNAc transferase but not G protein). Here we present a detailed characterization of the conditions required to achieve transport in vitro. We find that donor and acceptor activities differ markedly in certain of their properties. The donor activity is inhibited by N-ethylmaleimide but the acceptor activity is resistant. Donor activity is unstable in the absence of ATP or the cytosol fraction; acceptor activity is much more stable. This asymmetry may reflect the vectorial nature of the underlying biochemistry of protein transport. Both donor and acceptor are trypsin-sensitive, implying a need for cytoplasmically oriented membrane proteins. Transport occurs only in a restricted range of close to physiological conditions. ATP is absolutely required, although as little as 1 microM is sufficient. Transport is inhibited by ATP-gamma-sulfate and vanadate, suggesting that ATP hydrolysis is needed. By contrast, ionophores that dissipate membrane potentials and proton gradients do not inhibit transport. Monensin was also without effect in the cell-free system.

Adenosine Triphosphate↗
PubMed1985

Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack.

Using monoclonal antibodies and electron microscopy, we have localized N-acetylglucosamine transferase I within the Golgi apparatus. This enzyme initiates the conversion of asparagine-linked oligosaccharides to the complex type. We have found that the enzyme is concentrated in the central (or medial) cisternae of the Golgi stack. Cisternae at the cis and trans ends of the Golgi complex appear to lack this protein. These experiments establish a function for the medial portion of the Golgi and imply that the Golgi is partitioned into at least three biochemically and morphologically distinct cisternal compartments.

Acetylglucosamine↗
PubMed1985

Retention of membrane proteins by the endoplasmic reticulum.

We have used a monoclonal antibody specific for a hydrocarbon-induced cytochrome P450 to localize, by electron microscopy, the epitope-specific cytochrome P450. The cytochrome was found in the rough and smooth endoplasmic reticulum (ER) and the nuclear envelope of hepatocytes. Significant quantities of cytochrome P450 were not found in Golgi stacks. We also could not find any evidence of Golgi-associated processing of the Asn-linked oligosaccharide chains of two well-characterized ER membrane glycoprotein enzymes (glucosidase II and hexose-6-phosphate dehydrogenase), or of the oligosaccharides attached to the bulk of the glycoproteins of the ER membrane. We conclude that these ER membrane proteins are efficiently retained during a process of highly selective export from this organelle.

Acetylglucosaminidase↗
PubMed1984

Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine.

Transport of the VSV-encoded glycoprotein (G protein) between successive compartments of the Golgi has been reconstituted in a cell-free system and is measured, in a rapid and sensitive new assay, by the coupled incorporation of 3H-N-acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported during mixed incubations from the "donor" compartment in Golgi from VSV-infected CHO clone 15B cells (missing a key Golgi GlcNAc transferase) to the next, successive "acceptor" compartment (containing the GlcNAc transferase) in Golgi from wild-type CHO cells. Golgi fractions used in this assay have been extensively purified, and account for all of the donor and acceptor activity in the cells. Together with several other lines of evidence, this indicates that the cell-free system is highly specific, measuring only transport between sequential compartments in the Golgi stack. Transport in vitro is almost as efficient as in the cell, and requires ATP and the cytosol fraction in addition to protein components on the cytoplasmic surface of the Golgi membranes.

Acetylglucosamine↗
PubMed1984

The glycoprotein that is transported between successive compartments of the Golgi in a cell-free system resides in stacks of cisternae.

Electron microscope autoradiography has been used to localize the glycoprotein transported between successive compartments of the Golgi in a cell-free system. Both donor and acceptor Golgi fractions contain stacks of cisternae, which remain as discrete populations even after prolonged incubations together. The glycosylated VSV G protein, having received 3H-GlcNAc residues following its transport in vitro, is entirely within the population of acceptor stacks from the uninfected wild-type Golgi population (those housing GlcNAc transferase l). Quantitation of 3H grains reveals between 6,000 and 12,000 molecules of G protein introduced into each acceptor cisterna as a result of transport in the cell-free system, amounting to approximately 5% of its total membrane protein. This represents about the full complement of transported protein normally contained in a Golgi cisterna in vivo. Transport in the cell-free system is efficient and specific, preserving the integrity of the Golgi stack and its individual cisternae.

Acetylglucosamine↗
PubMed1984

Sequential intermediates in the pathway of intercompartmental transport in a cell-free system.

Two-stage incubations and the selective inhibitory effects of N-ethylmaleimide have revealed three steps in the transport of the vesicular stomatitis viral glycoprotein (G protein) between compartments of the Golgi. These are "priming" of the donor membrane, making G protein available for transfer to the acceptor Golgi stack; "transfer" of G protein to the acceptor stack to form a prefusion complex in which G protein is still separate from the GlcNAc transferase; and "fusion," the steps that result in the delivery of G protein to the same cisternal membranes that contain the GlcNAc transferase. Electron microscopy shows that priming of the donor membrane is accompanied by the formation of a uniform population of small (60-80 nm diameter) vesicles that bud from the rims of the cisternae of the Golgi stacks. This suggests the working hypothesis that the above steps correspond to stages in the budding and fusion of transport vesicles.

Adenosine Triphosphate↗
PubMed1984

Transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system.

Mixed monolayers containing vesicular stomatitis virus-infected Chinese hamster ovary clone 15B cells (lacking UDP-N-acetylglucosamine transferase I, a Golgi enzyme) and uninfected wild-type Chinese hamster ovary cells were formed. Extensive cell fusion occurs after the monolayer is exposed to a pH of 5.0. The vesicular stomatitis virus encoded membrane glycoprotein (G protein) resident in the rough endoplasmic reticulum (labeled with [35S]methionine) or Golgi complex (labeled with [3H]palmitate) of 15B cells at the time of fusion can reach Golgi complexes from wild-type cells after fusion; G protein present in the plasma membrane cannot. Transfer to wild-type Golgi complexes is monitored by the conversion of G protein to an endoglycosidase H-resistant form upon arrival, and also demonstrated by immunofluorescence microscopy. G protein in the Golgi complex of the 15B cells at the time of fusion exhibits properties vis a vis its transfer to an exogenous Golgi population identical to those found earlier in a cell-free system (Fries, E., and J. E. Rothman. 1981. J. Cell Biol., 90: 697-704). Specifically, pulse-chase experiments using the in vivo fusion and in vitro assays reveal the same two populations of G protein in the Golgi complex. The first population, consisting of G protein molecules that have just received their fatty acid, can transfer to a second Golgi population in vivo and in vitro. The second population, entered by G protein approximately 5 min after its acylation, is unavailable for this transfer, in vivo and in vitro. Presumably, this second population consists of those G-protein molecules that had already been transferred between compartments within the 15B Golgi population, in an equivalent process before cell fusion or homogenization for in vitro assays. Evidently, the same compartment boundary in the Golgi complex is detected by these two measurements. The surprisingly facile process of glycoprotein transit between Golgi stacks that occurs in vivo may therefore be retained in vitro, providing a basis for the cell-free system.

Acetylglucosamine↗
PubMed1984

Intercompartmental transport in the Golgi complex is a dissociative process: facile transfer of membrane protein between two Golgi populations.

The transfer of the vesicular stomatitis virus-encoded glycoprotein (G protein) between Golgi populations in fused cells (Rothman, J. E., L. J. Urbani, and R. Brands. 1984. J. Cell Biol. 99:248-259) is exploited here to study and to help define the compartmental organization of the Golgi stack and to characterize the mechanism of intercompartmental transport. We find that G protein that has just received its peripheral N-acetylglucosamine in the Golgi complex of one cell is efficiently transferred to the Golgi complex of another cell to receive galactose (Gal). Remarkably, this transport occurs at the same rate between these two compartments whether they are present in the same or different Golgi populations. Therefore, a dissociative (presumably vesicular) transport step moves G protein from one part of the Golgi in which N-acetylglucosamine is added to another in which Gal is added. Minutes later, upon receiving Gal, the same G protein molecules are very poorly transferred to an exogenous Golgi population after cell fusion. Therefore, once this intercompartmental transfer has already taken place (before fusion), it cannot take place again (after fusion); i.e., transport across the compartment boundary in the Golgi complex that separates the sites of N-acetylglucosamine and Gal incorporation is a vectorial process. We conclude that transfers between Golgi cisternae occur by a stochastic process in which transport vesicles budding from cisternae dissociate, can diffuse away, and then attach to and fuse with the appropriate target cisterna residing in the same or in a different stack, based on a biochemical pairing after a random encounter. Under these circumstances, a transported protein would almost always randomize among stacks with each intercisternal transfer; it would not progress systematically through a single stack. Altogether, our studies define three sequential compartments in the Golgi stack.

Acetylglucosamine↗
PubMed1984

An enzyme that removes clathrin coats: purification of an uncoating ATPase.

Uncoating ATPase, an abundant 70,000-mol-wt polypeptide mediating the ATP-dependent dissociation of clathrin from coated vesicles and empty clathrin cages, has been purified to virtual homogeneity from calf brain cytosol. Uncoating protein is present in cells in amounts roughly stoichiometric with clathrin. This enzyme is isolated as a mixture of monomers and dimers, both forms being active. ATP can support protein-facilitated dissociation of clathrin at micromolar levels; all other ribotriphosphates as well as deoxy-ATP are inactive. The clathrin that is released from cages consists of trimers (triskelions) in a stoichiometric complex with uncoating ATPase. These complexes with clathrin have little tendency to self-associate at neutral pH, and at acidic pH they interfere with the assembly of free clathrin. The possible existence and function of these complexes as clathrin carriers in cells would explain why uncoating protein is made in quantities equivalent to clathrin.

Adenosine Triphosphatases↗
PubMed1984

Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

Adenosine Triphosphatases↗