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Biomedical subjects

J E Rothman

Publications and source records attributed to J E Rothman.

At least 109 records · Page 6Linked to original sources
PubMed1991

A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin.

Four high-molecular-weight proteins form the main subunits of the coat of Golgi-derived (non-clathrin) coated vesicles. One of these coat proteins, beta-COP, is identical to a Golgi-associated protein of relative mass 110,000 (110K) that shares homology with the adaptin proteins of clathrin-coated vesicles. This connection, and the comparable molecular weights of the coat proteins of Golgi-derived and clathrin-coated vesicles, indicates that they may be structurally related. The identification of beta-COP as the 110K protein explains the blocking of secretion by the drug brefeldin A.

Adaptor Protein Complex beta Subunits↗
PubMed1991

'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles.

Golgi-derived coated vesicles contain a set of coat proteins of relative molecular mass 160,000 (Mr 160K; alpha-COP), 110K (beta-COP), 98K (gamma-COP) and 61K (delta-COP), and several smaller subunits. We have now identified and purified a cytosolic complex containing the same four coat proteins as those of Golgi transport vesicles. We term this complex the Golgi coat promoter or 'coatomer'. The coatomer also contains polypeptides of Mr 36K, 35K and 20K. It represents about 0.2% of soluble cytosolic protein. Gel filtration of unfractionated cytosol indicates that beta-COP resides exclusively in the coatomer complex. The complex seems to be a likely candidate for the unassembled precursor of Golgi coated vesicles, and its purification should help investigations of the role of coat proteins in membrane budding, for which it is necessary to use a refined cell-free system.

Adaptor Protein Complex beta Subunits↗
PubMed1991

Proteins involved in vesicular transport and membrane fusion.

In the past year, new information about proteins involved in vesicular transport has been plentiful. Particularly noteworthy are the complementary findings that Sec17p is required for vesicle consumption in endoplasmic reticulum-to-Golgi transport in yeast and that an analogous activity in mammalian cells, termed SNAP, is required for transport from the cis to the medial cisternae of the Golgi apparatus.

Animals↗
PubMed1991

Intracellular membrane fusion.

Protein trafficking and membrane assembly are accomplished in eukaryotes by the specific targeting and fusion of vesicles. In this review we describe some of the molecules implicated as components of the fusion apparatus, and evidence that suggests the same factors are recruited for a variety of intracellular fusion events.

Carrier Proteins↗
PubMed1990

Reconstitution of steps in the constitutive secretory pathway in permeabilized cells. Secretion of glycosylated tripeptide and truncated sphingomyelin.

The constitutive secretory pathway has been reconstituted in mechanically permeabilized Chinese hamster ovary cells using two secretory markers, an acyltripeptide (N-octanoyl-Asn-Tyr-Thr-NH2) that is glycosylated at Asn in the endoplasmic reticulum and a truncated ceramide that is converted to sphingomyelin. Secretion of these bulk phase markers is dependent on cytosolic proteins and ATP. Secretion of both the glycosylated tripeptide and truncated sphingomyelin was inhibited at 15 degrees C. These results are taken as evidence that the vesicle flow to the plasma membrane (rather than artificial lysis of endoplasmic reticulum or Golgi cisternae) is required for the release of markers to the medium. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a nonhydrolyzable analogue of GTP, inhibited secretion, resulting in an accumulation of both the glycosylated tripeptide and truncated sphingomyelin in the semi-intact cell. Inhibition of secretion by GTP gamma S was not observed in the presence of the aminoglycoside antibiotic neomycin.

Adenosine Triphosphate↗
PubMed1990

The rate of bulk flow from the Golgi to the plasma membrane.

A truncated analog of the backbone of sphingomyelin and glycolipids was synthesized. This truncated C8C8 ceramide was soluble in water (but was still able to cross cell membranes) and was utilized by the Golgi apparatus of living cells to produce water-soluble truncated phospholipids and glycolipids that were then secreted into the medium. Sphingomyelin is synthesized in a proximal (likely the cis) Golgi compartment. At 37 degrees C in CHO cells, the sphingomyelin analog is secreted with a half time of about 10 min. With this rate of bulk flow, no special signal is needed to pass through the Golgi to the plasma membrane. At 30 degrees C the half time of secretion of a lumenal ER marker is about 18 min, and that of the truncated sphingomyelin is about 14 min. Comparison of these rates sets an upper limit of about 4 min for half of the ER to be drained into the proximal Golgi at 30 degrees C.

Animals↗
PubMed1990

Purification of three related peripheral membrane proteins needed for vesicular transport.

We report conditions under which Golgi membranes depleted of peripheral membrane proteins can be reconstituted for intra-cisternal vesicular transport. Analysis of the reconstitution reveals requirements for N-ethylmaleimide-sensitive fusion protein, a purified peripheral protein involved in the fusion stage of vesicular transport, as well as other peripheral protein activities which can be provided by mammalian cytosol but not yeast cytosol. The restorative activity in bovine brain cytosol is found in two broad and complementing fractions, of average native molecular masses of about 500 and 40 kDa, termed Fr1 and Fr2, respectively. This resolved transport system was used to develop a purification scheme for Fr2. Three proteins of apparent molecular masses of 35, 36, and 39 kDa (Fr2-alpha, -beta, and -gamma, respectively) were found to be responsible for Fr2 activity and were purified to homogeneity. Each Fr2 protein has activity by itself in the reconstituted in vitro Golgi transport assay, although each exhibits a different specific activity and plateau value. No synergy of the three Fr2 proteins was observed during mixing experiments. The three Fr2 proteins seem to be closely related based on size, in vitro activities, chromatographic properties, and peptide maps and may comprise a new family of proteins involved in vesicular transport.

Animals↗
PubMed1990

SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast.

Three new and likely related components of the cellular fusion machinery have been purified from bovine brain cytosol, termed alpha-SNAP (35 kd), beta-SNAP (36 kd), and gamma-SNAP (39 kd). Transport between cisternae of the Golgi complex measured in vitro requires SNAP activity during the membrane fusion stage, and each SNAP is capable of binding the general cellular fusion protein NSF to Golgi membranes. The SNAP-NSF-membrane complex may be an early stage in the assembly of a proposed multisubunit "fusion machine" on the target membrane. SNAP transport factor activity is also found in yeast. Yeast cytosol prepared from a secretion mutant defective in export from the endoplasmic reticulum (sec17) lacks SNAP activity, which can be restored in vitro by the addition of pure alpha-SNAP, but not beta- or gamma-SNAPs. These data suggest that the mechanism of action of SNAPs in membrane fusion is conserved in evolution.

Animals↗
PubMed1990

Fatty acylation promotes fusion of transport vesicles with Golgi cisternae.

Two different methods, stimulation of transport by fatty acyl-coenzyme A (CoA) and inhibition of transport by a nonhydrolyzable analogue of palmitoyl-CoA, reveal that fatty acylation is required to promote fusion of transport vesicles with Golgi cisternae. Specifically, fatty acyl-CoA is needed after the attachment of coated vesicles and subsequent uncoating of the vesicles, and after the binding of the NEM-sensitive fusion protein (NSF) to the membranes, but before the actual fusion event. We therefore suggest that an acylated transport component participates, directly or indirectly, in membrane fusion.

Acyl Coenzyme A↗
PubMed1990

Movement of proteins through the Golgi stack: a molecular dissection of vesicular transport.

A combination of cell-free biochemical and morphological studies has revealed that a coated bud-coated vesicle transport system shuttles newly synthesized proteins through the successive processing compartments of the Golgi apparatus. These Golgi-coated vesicles operate in a manner formally analogous to the clathrin-coated, pit-coated vesicle system responsible for receptor-mediated endocytosis; however Golgi-coated vesicles do not contain clathrin.

Animals↗
PubMed1989

Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells.

Subcellular compartments in which folding and assembly of proteins occur seem to have a set of PCB proteins capable of mediating these and related processes, such as translocation across membranes. When a domain of a polypeptide chain emerges from a ribosome during synthesis or from the distal side of a membrane during translocation, successive segments of the chain are incrementally exposed to solvent and yet are unlikely to be able to fold. This topological restriction on folding likely mandates a need for PCB proteins to prevent aggregation. Catalysis of topologically restricted folding by PCB proteins is likely to involve both an antifolding activity that postpones folding until entire domains are available and, more speculatively, a folding activity resulting from a programmed stepwise release that employs the energy of ATP hydrolysis to ensure a favorable pathway. We are left with a new set of problems. How do proteins fold in cells? What are the sequences or structural signals that dictate folding pathways? The new challenge will be to understand folding as a combination of physical chemistry, enzymology, and cell biology.

Animals↗
PubMed1989

Fatty acyl-coenzyme A is required for budding of transport vesicles from Golgi cisternae.

We describe a new role for fatty acylation. Conditions were established under which vesicular transport from the cis to the medial Golgi compartment in vitro depends strongly upon the addition of a fatty acyl-coenzyme A, e.g., palmitoyl-CoA. Using an inhibitor of long-chain acyl-CoA synthetase, we demonstrate that the fatty acid has to be activated by CoA to stimulate transport. A nonhydrolyzable analog of palmitoyl-CoA competitively inhibits transport. Electron microscopy and biochemical studies show that fatty acyl-CoA is required for budding of (non-clathrin-) coated transport vesicles from Golgi cisternae and that budding is inhibited by the nonhydrolyzable analog.

Acyl Coenzyme A↗
PubMed1989

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack.

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.

Animals↗
PubMed1989

Peptide binding and release by proteins implicated as catalysts of protein assembly.

Two members of the hsp70 family, termed hsc70 and BiP, have been implicated in promoting protein folding and assembly processes in the cytoplasm and the lumen of the endoplasmic reticulum, respectively. Short hydrophilic (8 to 25 residues) synthetic peptides have now been tested as possible mimics of polypeptide chain substrates to help define an enzymatic basis for these activities. Both BiP and hsc70 have specific peptide binding sites. Peptide binding elicits hydrolysis of adenosine triphosphate, with the subsequent release of bound peptide.

Adenosine Triphosphate↗
PubMed1989

A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast.

A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.

Amino Acid Sequence↗
PubMed1989

Vesicular transport between the endoplasmic reticulum and the Golgi stack requires the NEM-sensitive fusion protein.

An N-ethylmaleimide-sensitive fusion protein (NSF) has been purified on the basis of its ability to catalyse vesicular transport within the Golgi stack. We report here that this same protein is required for transport from the endoplasmic reticulum to the Golgi stack in semi-intact cells. This transport process is inhibited by a monoclonal antibody against NSF. Furthermore, pretreatment of semi-intact cells with N-ethylmaleimide, a sulphydryl alkylating reagent, inhibits transport. Addition of highly purified NSF largely restores transport from endoplasmic reticulum to Golgi. These results suggest that NSF is a general component of the transport machinery required for membrane fusion at multiple stages of the secretory pathway.

Animals↗