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Biomedical subjects

J E Rothman

Publications and source records attributed to J E Rothman.

At least 55 records · Page 3Linked to original sources
PubMed1996

Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25.

Synaptotagmin, a likely calcium sensor for synaptic transmission, and SNAP-25, a t-SNARE of the presynaptic plasma membrane, are key proteins for the docking and fusion of synaptic and other vesicles. We report that both synaptotagmin and SNAP-25 are palmitoylated with their fatty acids attached in a labile thioester-type bond. A SNAP-25 mutant with deleted palmitoylation sites is found exclusively in the cytosol after cell fractionation, whereas the palmitoylated form of SNAP-25 is membrane-bound, establishing that SNAP-25 is membrane-anchored via covalently linked palmitate.

Acylation↗
PubMed1996

Protein sorting by transport vesicles.

Eukaryotic life depends on the spatial and temporal organization of cellular membrane systems. Recent advances in understanding the machinery of vesicle transport have established general principles that underlie a broad variety of physiological processes, including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled release of hormones and neurotransmitters.

Amino Acid Sequence↗
PubMed1996

The protein machinery of vesicle budding and fusion.

A general protein machinery that buds and fuses transport vesicles is harnessed to generate the complex web of intracellular transport pathways critical for such diverse processes as cell growth, endocytosis, hormone release, and neurotransmission. With this appreciation, the challenge of understanding the precise molecular mechanisms of these many facets of cell biology has been reduced to a series of problems in protein structure and chemistry.

ADP-Ribosylation Factors↗
PubMed1996

A v-SNARE implicated in intra-Golgi transport.

We report the identification of a putative v-SNARE (GOS-28), localized primarily to transport vesicles at the terminal rims of Golgi stacks. In vitro, GOS-28, A Golgi SNARE of 28 kD, is efficiently packaged into Golgi-derived vesicles, which are most likely COPI coated. Antibodies directed against GOS-28 block its ability to bind alpha-SNAP, partially inhibit transport from the cis to the medial cisternae, and do not inhibit budding of COP-coated vesicles, but do accumulate docked uncoated vesicles.

Amino Acid Sequence↗
PubMed1996

Molecular machinery mediating vesicle budding, docking and fusion.

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.

Animals↗
PubMed1995

A possible docking and fusion particle for synaptic transmission.

Several proteins have been implicated in the rapid (millisecond) calcium-controlled release of transmitters at nerve endings, including soluble N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (alpha-SNAP), the synaptic SNAP receptor (SNARE) and the calcium-binding protein synaptotagmin, which may function as a calcium sensor in exocytosis. A second SNAP isoform (beta-SNAP), which is 83% identical to alpha-SNAP, is highly expressed in brain, but its role is still unclear. Here we show that these proteins assemble cooperatively to form a docking and fusion complex. beta-SNAP (but not alpha-SNAP) binds synaptotagmin and recruits NSF, indicating that the complex may link the process of membrane fusion to calcium entry by attaching a specialized fusion protein (beta-SNAP) to a calcium sensor (synaptotagmin). Polyphosphoinositols that block transmitter release, inositol 1,3,4,5-tetrakisphosphate (InsP4), inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), also block the assembly of the particle by preventing beta-SNAP from binding to synaptotagmin.

Calcium↗
PubMed1995

An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.

ADP-Ribosylation Factors↗
PubMed1995

SNAP-mediated protein-protein interactions essential for neurotransmitter release.

The constitutive fusion of transport vesicles with intracellular membranes requires soluble proteins called SNAPs. Certain presynaptic proteins implicated in synaptic vesicle exocytosis also bind SNAPs, suggesting that SNAPs participate in the calcium-regulated membrane fusion events mediating neurotransmitter release. Here we show that injection of recombinant SNAPs into the giant synapse of squid enhances transmitter release. Conversely, injection of peptides designed to mimic the sites at which SNAP interacts with its binding partners inhibits transmitter release downstream of synaptic vesicle docking. A SNAP-dependent protein complex must therefore mediate transmitter release, showing that transmitter release shares a common molecular mechanism with constitutive membrane fusion.

Amino Acid Sequence↗
PubMed1995

The capacity to retrieve escaped ER proteins extends to the trans-most cisterna of the Golgi stack.

To explore how far into the Golgi stack the capacity to retrieve KDEL proteins extends, we have introduced an exogenous probe (the peptide YHPNSTCSEKDEL) into the TGN of living cells. For this purpose, a CHO cell line expressing a c-myc-tagged version of the transmembrane protein TGN38--which cycles between the TGN and the cell surface--was generated. The cells internalized peptides that were disulfide bonded to anti-myc antibodies and accumulated the peptide-antibody complexes in the TGN. Peptides released from these complexes underwent retrograde transport to the ER, as evidenced by the transfer of N-linked carbohydrate to their acceptor site. The KDEL-tagged glycopeptides (approximately 10% of the endocytosed load) behaved like endogenous ER residents: they stayed intracellular, and their oligosaccharide side chains remained sensitive to endoglycosidase H. An option thus exists to extract ER residents even at the most distant pole of the Golgi stack, suggesting that sorting of resident from exported ER proteins may occur in a multistage process akin to fractional distillation.

Amino Acid Sequence↗
PubMed1994

Coatomer-rich endoplasmic reticulum.

We identify in normal cells the existence of two distinct sites of the transitional endoplasmic reticulum (ER), one housing the Sec23p protein complex (the classical transitional element), the other the coatomer protein complex (the coatomer-rich ER). Experimental conditions that reduce transport from the ER to the Golgi complex lead to the overexpression of this newly defined coatomer-rich ER.

Animals↗
PubMed1994

Mechanisms of intracellular protein transport.

Recent advances have uncovered the general protein apparatus used by all eukaryotes for intracellular transport, including secretion and endocytosis, and for triggered exocytosis of hormones and neurotransmitters. Membranes are shaped into vesicles by cytoplasmic coats which then dissociate upon GTP hydrolysis. Both vesicles and their acceptor membranes carry targeting proteins which interact specifically to initiate docking. A general apparatus then assembles at the docking site and fuses the vesicle with its target.

Animals↗
PubMed1994

A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles.

Rab proteins are generally required for transport vesicle docking. We have exploited yeast secretion mutants to demonstrate that a rab protein is required for v-SNAREs and t-SNAREs to assemble. The absence of the rab protein in the docking complex suggests that, in a broad sense, rab proteins participate in a reaction catalyzing SNARE complex assembly. In so doing, rab proteins could help impart an additional layer of specificity to vesicle docking. This mechanism likely involves the Sec1 homolog Sly1, which we identified in isolated docking complexes. We also report the identification of a novel v-SNARE (Ykt6p) component of the yeast ER-Golgi docking complex that has a CAAX box and is predicted to be lipid anchored. The surprising finding that docking complexes can contain many distinct species of SNAREs (Sed5p, Bos1p, Sec22p, Ykt6p, and likely Bet1p, p28, and p14) suggests that multimeric interactions are features of the fusion machinery, and may also improve the fidelity of vesicle targeting.

Amino Acid Sequence↗
PubMed1994

Implications of the SNARE hypothesis for intracellular membrane topology and dynamics.

The SNARE hypothesis provides a mechanism for the specific docking and fusion of transport vesicles with their target membranes. A simple extension of the hypothesis can explain many cellular processes, including the stacking of Golgi cisternae, retrograde transport and homotypic fusion; it can also explain the morphology of intracellular membranes and their dynamics during mitosis.

Animals↗
PubMed1994

Removal of Rab GTP-binding proteins from Golgi membranes by GDP dissociation inhibitor inhibits inter-cisternal transport in the Golgi stacks.

Rab proteins are a family of Ras-like GTPases involved in intracellular membrane traffic. Rab GDI, a cytosolic protein which inhibits the dissociation of GDP from various Rab proteins, is required to maintain a pool of Rab proteins in the cytosol. We describe the purification of a cytosolic factor from bovine liver that inhibits intra-cisternal transport between the Golgi stacks. We identify this factor as a Rab GDI. Half-maximal inhibition of transport was observed in the presence of the same concentration of GDI that is required for removal of Rab proteins from the Golgi.

Amino Acid Sequence↗
PubMed1994

Neurotransmission: harnessing fusion machinery at the synapse.

Neurotransmission requires the docking of synaptic vesicles to the presynaptic plasma membrane, and their signal-dependent fusion. These processes use a general 'machinery' operating at several intracellular vesicular transport steps and, in addition, use a set of unique components that characterizes this specific form of regulated secretion. This review summarizes recent progress that has significantly increased our understanding of how intracellular transport vesicles dock and fuse with their target membrane, both in the synapse and elsewhere.

Animals↗
PubMed1994

Intracellular membrane fusion.

The NSF, SNAP, and SNAP receptors are key elements of the intracellular membrane fusion machinery. We use an affinity purification scheme, based on the function of SNAP receptor in assembling 20S fusion particles from NSF and SNAP proteins, to purify SNAP receptors from brain. Remarkably, each of the four SNAP receptors (or, SNAREs) thus delineated resides in synapses, with one receptor originating in the synaptic vesicle and another in the presynaptic plasma membrane that is targeted for fusion. This suggests a simple mechanism in which the general NSF/SNAP fusion machinery can assemble to bridge partner membranes in a complex containing elements of both vesicle and target membranes, and implies that similar fusion machines drive both constitutive fusion (ER-->Golgi-->surface and endocytosis) and regulated exocytosis. The vesicle (v-SNARE) and the target-associated t-SNAREs from the synapse are each members of compartmentally-specific families of membrane proteins found in yeast, animal cells, and neurons, thus raising the possibility that v-SNAREs and t-SNAREs encode specificity in membrane fusion processes that utilize a common mechanism.

Amino Acid Sequence↗
PubMed1994

ADP-ribosylation factor and coatomer couple fusion to vesicle budding.

The coat proteins required for budding COP-coated vesicles from Golgi membranes, coatomer and ADP-ribosylation factor (ARF) protein, are shown to be required to reconstitute the orderly process of transport between Golgi cisternae in which fusion of transport vesicles begins only after budding ends. When either coat protein is omitted, fusion is uncoupled from budding-donor and acceptor compartments pair directly without an intervening vesicle. Coupling may therefore results from the sequestration of fusogenic membrane proteins into assembling coated vesicles that are only exposed when the coat is removed after budding is complete. This mechanism of coupling explains the phenomenon of "retrograde transport" triggered by uncouplers such as the drug brefeldin A.

ADP-Ribosylation Factors↗