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Biomedical subjects

J E Rothman

Publications and source records attributed to J E Rothman.

At least 181 records · Page 10Linked to original sources
PubMed1977

Synchronised transmembrane insertion and glycosylation of a nascent membrane protein.

Studies of the synthesis and incorporation of the vesicular stomatitis virus glycoprotein into membranes in a synchronised cell-free system demonstrate a tight coupling between polypeptide synthesis and membrane insertion, as a result of which the nascent chain crosses the membrane. The studies reveal a surprisingly precise sequence by which the nascent chain of this membrane glycoprotein is glycosylated in two steps. These findings have important implications for the mechanisms of membrane assembly.

Cell-Free System↗
PubMed1977

Membrane asymmetry.

The components of biological membranes are asymmetrically distributed between the membrane surfaces. Proteins are absolutely asymmetrical in that every copy of a polypeptide chain has the same orientation in the membrane, and lipids are nonabsolutely asymmetrical in that almost every type of lipid is present on both sides of the bilayer, but in different and highly variable amounts. Asymmetry is maintained by lack of transmembrane diffusion. Two types of membrane proteins, called ectoproteins and endoproteins, are distinguished. Biosynthetic pathways for both types of proteins and for membrane lipids are inferred from their topography and distribution in the formed cells. Note added in proof. A cell-free system has now been developed which permits the mechanisms of membrane protein assembly to be studied (108). The membrane glycoprotein of vesicular stomatitis virus has been synthesized by wheat germ ribosomes in the presence of rough endoplasmic reticulum from pancreas. The resulting polypeptide is incorporated into the membrane, spans the lipid bilayer asymmetrically, and is glycosylated (108). The amino terminal portion of this transmembrane protein is found inside the endoplasmic reticulum vesicle, while the carboxyl terminal portion is exposed on the outer surface of the vesicle. Furthermore, addition of the glycoprotein to membranes after protein synthesis does not result in incorporation of the protein into the membrane in the manner described above (108). Consequently, protein synthesis and incorporation into the membrane must be closely coupled. Indeed, using techniques to synchronize the growth of nascent polypeptides, it has been shown (109) that no more than one-fourth of the glycoprotein chain can be made in the absence of membranes and still cross the lipid bilayer when chains are subsequently completed in the presence of membranes. These findings demonstrate directly that the extracytoplasmic portion of an ectoprotein can cross the membrane only during biosynthesis, and not after.

Animals↗
PubMed1977

Membrane assembly: synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein.

The glycoprotein (G) of vesicular stomatitis virus (VSV) is synthesized on membrane-bound polyribosomes. Approximately 30 min after its synthesis, it reaches the surface plasma membrane where it is incorporated into budding virus. The first part of this paper focuses on the 2 intracellular, membrane-bound, glycosylated forms of the glycoprotein which are intermediates in its biogenesis. All glycosylation and processing is completed in the smooth microsome fraction before the protein reaches the surface. Next, we turn to the mechanism by which G is synthesized on membrane-bound polyribosomes. All of the G mRNA is bound to membranes, and studies with puromycin suggest that this attachment of G mRNA is mediated by the nascent glycoprotein chain. After its synthesis G is a transmembrane protein with about 30 amino acids at the carboxyl terminus remaining on the cytoplasmic side of the endoplasmic reticulum. Since 95% of the glycoprotein, containing the carbohydrate residues, is resistant to attack by external proteases, it appears to be within the lumen of the endoplasmic reticulum or embedded within the lipid bilayer. Finally, we show that synthesis, glycosylation, and proper asymmetric insertion of G into the ER can be achieved in cell-free extracts. Both glycosylation of G and proper insertion into the ER membrane in this cell-free system require concomitant protein synthesis.

Cell Line↗
PubMed1977

Rapid transmembrane movement of newly synthesized phospholipids during membrane assembly.

The transbilayer distribution of phospholipids in Bacillus megaterium is asymmetrical, with twice as much phosphatidylethanolamine internally as externally (Rothaman, J. E. & Kennedy, E. P. (1977) J. Mol. Biol. 110,603-618). We now report that the biosynthesis of phosphatidylethanolamine is also asymmetrical. Newly synthesized phosphatidylethanolamine was found first on the cytoplasmic side of the membrane of pulse-labeled cells and later was redistributed until the specific radioactivity of the outer face became equal to that of the inner face of the bilayer. The rate of transmembrane movement is at least 30,000 times faster than the rate of spontaneous diffusion (flip-flop) of phosphatidylethanolamine across artificial phospholipid bilayers, indicating that transmembrane movement must be a facilitated process in living cells, perhaps involving membrane proteins.

Bacillus megaterium↗
PubMed1977

Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein.

Membrane assembly was observed to proceed in cell-free extracts. Specifically, the membrane glycoprotein of vesicular stomatitis virus was synthesized in crude extracts of wheat germ in the presence of membrane vesicles derived from pancreatic endoplasmic reticulum. The resulting glycoprotein spans the lipid bilayer asymmetrically, is glycosylated, and is indistinguishable in these respects from the form of the glycoprotein found in the rough endoplasmic reticulum of virus-infected cells. Both glycosylation and asymmetric transmembrane insertion of the glycoprotein into membranes in vitro require protein synthesis in the presence of membranes. The carboxyl-terminal 5% of the polypeptide chain is located on the external surface of vesicles, corresponding to the cytoplasmic surface of the endoplasmic reticulum in cells. Most, or all, of the amino-terminal portion of the glycoprotein, as well as the protein-bound carbohydrate, appears to be located within the lumen of the membrane vesicles. These findings demonstrate that insertion of this membrane protein occurs during or immediately after protein synthesis. The results are consistent with the concepts that the growing membrane protein is extruded across the endoplasmic reticulum membrane amino terminus first and that glycosylation is restricted to the lumenal surface of the membrane. The cell-free system reported here should prove valuable for studying these processes.

Animals↗
PubMed1976

Fusion and protein-mediated phospholipid exchange studied with single bilayer phosphatidylcholine vesicles of different density.

A novel method has been developed for the study of phospholipid exchange and fusion of phospholipid vesicles. Two homogeneous populations of single bilayer phosphatidylcholine vesicles of similar size but markedly different density have been prepared. "Dense" vesicles were made from brominated dioleoyl phosphatidylcholine. "Light" vesicles were prepared from dioleoyl phosphatidylcholine. The two populations were easily separated by density gradient centrifugation. Phosphatidylcholine exchange protein from beef liver was used to promote lecithin exchange between the vesicle populations. Only the lecithin of the external monolayers of the vesicles was available for exchange by exchange protein, implying that flip-flop of vesicle phosphatidylcholine did not take place at a detectable frequency. No spontaneous intervesicle phosphatidylcholine exchange was observed. However, the dense and light vesicles did spontaneously fuse, over several hours, to produce particles of hybrid density.

Biological Transport↗
PubMed1976

Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus.

Two phospholipid exchange proteins and two phospholipases C have been employed to determine the phospholipid composition of the outer surface of the membrane of influenza virus. These four protein probes have defined the same accessible and inaccessible pool for each viral phospholipid. Phospholipids which are exchangeable or hydrolyzable are located on the outer surface, whereas the inaccessible pool is located at the inner surface of the viral bilayer. The two pools are unequal in size, with ca. 30% of the total phospholipid accessible to the four proteins, and ca. 70% inaccessible. The membrane is thus highly asymmetric with regard to the amount of phospholipid on each side of the membrane. There is also a marked asymmetry of phospholipid composition. Phosphatidylcholine and phosphatidylinositol are enriched in the outer surface, and sphingomyelim is enriched in the inner surface, whereas phosphatidylethanolamine and phosphatidylserine are present in similar proportions in each surface. This distribution is qualitatively different from that previously reported for the human erythrocyte. The close agreement between results obtained with excahnge proteins and phospholipases C demonstrates that the hydrolytic action of these enzymes does not alter phospholipid asymmetry. The nonperturbing nature of the exchange proteins has permitted the rate of transmembrane movement of phospholipids (flip-flop) in the intact virion to be studied. This process could not be detected after 2 days at 37 degrees C. It was estimated that the half-time for flip-flop is indeterminately in excess of 30 days for sphingomyelin and 10 days for phosphatidylcholine at 37 degrees C. These extremely long times provide a simple explanation for the maintenance of transbilayer asymmetry in influenza virions and possibly, other membranes. Since the viral membrane is acquired by budding through the host cell plasma membrane, the transbilayer distribution of phospholipids observed in the virions presumably reflects a similar asymmetric distribution of phospholipids in the host cell surface membrane. Because animal cells in culture do not incorporate extracellular phospholipid, our results demonstrate that individual cells have the capacity to generate asymmetric membranes.

Binding Sites↗
PubMed1976

Transbilayer distribution and movement of cholesterol and phospholipid in the membrane of influenza virus.

The transfer of radioactive cholesterol from influenza virus to excess phosphatidylcholine-cholesterol vesicles has been studied. Viral cholesterol was found to exist in two pools, one rapidly exchangeable. Evidence is presented that the rapidly exchangeable pool corresponds to cholesterol present on the outer surface of the viral bilayer, while the slowly exchangeable pool corresponds to inner surface cholesterol. Approximately equal amounts are present in each pool, suggesting that cholesterol distribution is not markedly asymmetric in the viral bilayer. A half-time for the rate of equilibration between the two sides of the bilayer (flip-flop) was about 13 days at 37 degrees with a 90% confidence interval of 3.4- infinity days. Similar experiments were carried out which followed the time course of transfer of labeled phospholipids from the viral bilayer to phospholipid vesicles, catalyzed by phospholipid exchange protein from beef heart. From these experiments the half-times for the flip-flop of phosphatidyl-choline and spingomyelin were found to be indeterminately in excess of 10 and 30 days at 37 degrees, respectively. These results suggest that exchange of the major components of the viral bilayer between the two surfaces occurs very slowly relative to the turnover times of most membrane constituents, and provide a plausible mechanism for the maintenance of membrane asymmetry over biologically relevant time periods.

Cholesterol↗
PubMed1975

Asymmetric exchange of vesicle phospholipids catalyzed by the phosphatidylcholine exhange protein. Measurement of inside--outside transitions.

Purified phosphatidylcholine exchange protein was used to exchange phosphatidylcholine between homogeneous single-walled phosphatidylcholine vesicles and human erythrocyte ghosts. When excess ghosts were present, it was found that only 70% of the vesicle phosphatidylcholine was available for exchange. This fraction corresponds closely to the amount of phosphatidycholine in the outer monolayer of these vesicles, indicating that only the outer surface of the vesicle is accessible to the exchange protein. Also, it was found that all phosphatidylcholine introduced into vesicles by the exchange protein was available for subsequent exchange. Using the exchange protein, asymmetrical vesicles were prepared in which the outer monolayer was either enriched or depleted in radioactive phosphatidylcholine as compared to the inner monolayer. Re-equilibration of the radioactivity between the two surfaces of the vesicle (flip-flop) could not be detected, even after 5 days at 37degrees. It is estimated that the half-time for flip-flop is in excess of 11 days at 37degrees. These results indicate that the properties of the exchange protein can be expolited to measure phosphatidylcholine flip-flop rates and possible phosphatidylcholine asymmetry in biological and model membranes, without altering the structure of the membrane.

Cell Membrane↗
PubMedDate not supplied

A role for clathrin light chains in the recognition of clathrin cages by 'uncoating ATPase'.

A cycle of clathrin assembly and disassembly drives the formation of coated vesicles, intermediates in intracellular protein transport. The heavy chain of clathrin is needed for assembly, but the function of the clathrin light chains has remained obscure. An enzyme has now been purified which uses ATP hydrolysis to power the release of clathrin from coated vesicles, presumably recycling the coat protein for repeated rounds of vesicle budding. This 'uncoating ATPase' requires clathrin light chains for its action.

Adenosine Triphosphatases↗
PubMedDate not supplied

Possible role for fatty acyl-coenzyme A in intracellular protein transport.

The transport of proteins between subcellular compartments is a vectorial, energy-requiring process mediated by the budding and fusion of a series of vesicular carriers. As yet, nothing is known of the chemical reactions that underlie these events, or how or in exactly what forms energy is used to sustain such movements. Here we report that fatty acyl-CoA acts as cofactor to a Golgi-associated protein factor (termed NSF) that is required for transport between cisternae of the Golgi stack in a cell-free system. This previously unsuspected connection may offer a link between the complex process of protein transport and a single, well-defined type of chemical reaction. We suggest that an ATP-dependent cycle of fatty acylation and deacylation may play an important role in driving rounds of vectorial protein transport.

Acyl Coenzyme A↗
PubMedDate not supplied

Felix Hoppe-Seyler Lecture 1996. Mechanisms of intracellular protein transport.

Discovery of the protein machinery of vesicle transport has followed the reconstitution of transport in a cell-free system, and established general mechanisms 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. Transport vesicles are formed as coat proteins assemble on membranes, are targetted by SNARE proteins, and fuse using a general mechanism involving SNAP proteins and the ATPase NSF.

Biological Transport↗