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W E Balch

Publications and source records attributed to W E Balch.

At least 109 records · Page 6Linked to original sources
PubMed1990

Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro.

Parallel experiments in living cells and in vitro were undertaken to characterize the mechanism by which misfolded and unassembled glycoproteins are retained in the ER. A thermoreversible folding mutant of vesicular stomatitis virus (VSV) G protein called ts045 was analyzed. At 39 degrees C, newly synthesized G failed to fold correctly according to several criteria: intrachain disulfide bonds were incomplete; the B2 epitope was absent; and the protein was associated with immunoglobulin heavy chain binding protein (BiP), a heat shock-related, ER protein. When the temperature was lowered to 32 degrees C, these properties were reversed, and the protein was transported to the cell surface. Upon the shift up from 32 degrees C back to 39 degrees C, G protein in the ER returned to the misfolded form and was retained, while the protein that had reached a pre-Golgi compartment or beyond was thermostable and remained transport competent. The misfolding reaction could be reconstituted in a cell free system using ts045 virus particles and protein extracts from microsomes. Taken together, the results showed that ER is unique among the organelles of the secretory pathway in containing specific factors capable of misfolding G protein at the nonpermissive temperature and thus participating in its retention.

Animals↗
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↗
PubMed1989

Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus.

Ca2+ and GTP hydrolysis are shown to be required for the transport of protein between the ER and the cis-Golgi compartment in semiintact cells, an in vitro system that reconstitutes transport between intact organelles. Transport was inhibited rapidly and irreversibly in the presence of micromolar concentrations of the nonhydrolyzable GTP analogue, GTP gamma S. The transport block in the presence of GTP gamma S was found to be distal to a post-ER, pre-Golgi compartment where proteins accumulate during incubation at 15 degrees C. In addition, transport was completely inhibited in the absence of free Ca2+. A sharp peak defining optimal transport between the ER and the cis-Golgi was found to occur in the presence of 0.1 microM free Ca2+. Inhibition of transport in the absence of free Ca2+ was found to be fully reversible allowing the step inhibited by GTP gamma S to be assigned to a position intermediate between the ER and the Ca2+ requiring step. The results suggest that GTP hydrolysis may trigger a switch to insure vectorial transport of protein along the ER/Golgi pathway, and that a free Ca2+ level similar to the physiological levels found in interphase cells is essential for a terminal step in vesicle delivery to the cis-Golgi compartment.

Aluminum↗
PubMed1987

Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex.

We introduce a new method that removes portions of the plasma membrane of eukaryotic cells to form semi-intact cells. During preparation, these cells lose their soluble cytoplasmic contents, but retain secretory organelles such as the ER and Golgi complex in an intact form. Transport of protein between the ER and Golgi can be functionally reconstituted in vitro using these semi-intact cells by incubation in the presence of cytosol and ATP. Export of the vesicular stomatitis virus strain tsO45 G protein from the ER in vitro is temperature-sensitive, similar to the result observed in vivo. These cells allow direct access of chemicals and antibodies to the cytoplasmic domain of the cell and may be a widely applicable model system for study of a broad range of problems in cell biology.

Adenosine Triphosphate↗
PubMed1987

Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system.

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.

Animals↗
PubMed1987

Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers.

We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.

Adenosine Triphosphate↗
PubMed1986

ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi.

The temperature and ATP dependence of transport of the vesicular stomatitis virus strain ts045 G protein from the endoplasmic reticulum (ER) to an early Golgi compartment containing mannosidase I was studied in the mutant Chinese hamster ovary cell clone 15B. Appearance of G protein containing the Man5GlcNAc2 oligosaccharide species occurred after a shift to the permissive temperature with a lag period of 5 min and without detectable formation of the intermediate Man7GlcNAc2 and Man6GlcNAc2 species. Two biochemically distinct transport steps were detected during transport from the ER to the Golgi. An initial step is temperature sensitive, thermoreversible, and requires a high threshold of cellular ATP for maximal rate of transport (80% of the normal cellular ATP pool). Export from the ER is inhibited at 65% of the normal cellular ATP pool. Prolonged incubation at reduced levels of cellular ATP or at the restrictive temperature resulted in the accumulation of G protein in either the Man8GlcNAc2 species or the Man7GlcNAc2 and Man6GlcNAc2 species, respectively. Reversal of the temperature-sensitive block is ATP coupled. A second step is insensitive to incubation at the restrictive temperature and proceeds efficiently when the cellular ATP pool is reduced to 20% of the control. G protein accumulates at this intermediate step during prolonged incubation at 15 degrees C. The data suggest a functional division of processes required for transport of protein between the ER and Golgi compartments. The two steps may reflect the export (budding) and delivery (fusion) of proteins through vesicular trafficking between the ER and Golgi.

Adenosine Triphosphate↗
PubMed1986

ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments.

The oligosaccharide processing intermediates of the vesicular stomatitis virus strain ts045 G protein were used to identify ATP- and temperature-sensitive steps in the constitutive pathway of protein transfer to the cell surface. In addition to the initial ATP-sensitive step required for export from the endoplasmic reticulum (Balch, W. E., Elliott, M. M., and Keller, D. S. (1986) J. Biol. Chem. 261, 14681-14689), two distinct ATP-sensitive steps functionally dissect the Golgi into at least 3 compartments: a cis compartment containing the trimming enzyme mannosidase I, a medial compartment conferring resistance to endoglycosidase H, and a trans compartment containing terminal glycosyl transferases. A fourth ATP-sensitive step is required for export of G protein from the trans Golgi to the cell surface. A high threshold of cellular ATP (70% of the control) was required for maximal rates of transport between Golgi compartments. Transport between compartments is inhibited at 40% of the normal cellular ATP pool. Only a single temperature-sensitive step localized to the endoplasmic reticulum inhibited transport of ts045 G protein to the cell surface. The data suggest that ATP-sensitive steps punctuate transport of protein between compartmental boundaries of the secretory pathway.

Adenosine Triphosphate↗
PubMed1986

A novel prefusion complex formed during protein transport between Golgi cisternae in a cell-free system.

Examination of a cell-free reconstitution of intercompartmental transport through the Golgi apparatus has enabled detection of two intermediates in the pathway (Balch, W. E., Glick, B. S., and Rothman, J. E. (1984) Cell 39, 525-536). These intermediates are thought to represent stages in the budding and fusion reactions of transport vesicles mediating such a transport process. Here we describe a new transport intermediate that is interposed between the previously established primed donor formation and the N-ethylmaleimide (NEM)-resistant acceptor intermediates. Consumption of this intermediate requires much less cytosol than its formation, and thus it has been termed the "dilution-resistant" intermediate. The dilution-resistant intermediate only forms in the presence of donor and acceptor membranes, and its consumption is sensitive to NEM. The transition from this state to the later, NEM-resistant form of the prefusion complex requires ATP as well as cytosol and may represent a processing of transport vesicles to permit their fusion.

Adenosine Triphosphate↗
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↗
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↗
PubMed1980

Sequence homologies in the N-terminal region of the ribosomal 'A' proteins from Methanobacterium Thermoautotrophicum and Halobacterium cutirubrum.

The ribosomal 'A' protein from the methanogen, Methanobacterium thermoautotrophicum, has been isolated and purified. The amino acid composition and mobility on two-dimensional gels indicates that this protein shows characteristics similar to the equivalent protein from the extreme halophile, Halobacterium cutirubrum, and is significantly different from the equivalent protein from other bacteria. The first 48 residues of the methanogenic 'A' protein were sequenced and showed a large amount of sequence homology to the H. cutirubrum 'A' protein. When the sequences of these two proteins were compared to the 'A' protein from other procaryotes (eubacteria), much less homology was evident. These results support the hypothesis (Woese, C.R. and Fox, G.F., (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5088-5090) of two procaryotic lines of evolutionary descent, the eubacteria and the archaebacteria, the latter including the methanogens and the extreme halophiles. The sequence data from the 'A' proteins also indicate that, phylogenetically, the archaebacteria are much closer to the cytoplasmic components of eucaryotes than they are to the eubacteria (or 'true bacteria').

Amino Acid Sequence↗