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1994 |
Baker, D; Agard, D A Kinetics versus thermodynamics in protein folding. Journal Article Biochemistry, 33 , pp. 7505-9, 1994, ISSN: 0006-2960. @article{571, title = {Kinetics versus thermodynamics in protein folding.}, author = { D Baker and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/kineticsversusthermodynamics_Baker1994.pdf}, issn = {0006-2960}, year = {1994}, date = {1994-06-01}, journal = {Biochemistry}, volume = {33}, pages = {7505-9}, abstract = {Until quite recently it has been generally believed that the observed tertiary structure of a protein is controlled by thermodynamic and not kinetic processes. In this essay we review several recent results which call into question the universality of the thermodynamic hypothesis and discuss their implications for the understanding of protein folding.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Until quite recently it has been generally believed that the observed tertiary structure of a protein is controlled by thermodynamic and not kinetic processes. In this essay we review several recent results which call into question the universality of the thermodynamic hypothesis and discuss their implications for the understanding of protein folding. |
1993 |
Baker, D; Shiau, A K; Agard, D A The role of pro regions in protein folding. Journal Article Current Opinion in Cell Biology, 5 , pp. 966-70, 1993, ISSN: 0955-0674. @article{570, title = {The role of pro regions in protein folding.}, author = { D Baker and A K Shiau and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/theroleofproregions_Baker1993.pdf}, issn = {0955-0674}, year = {1993}, date = {1993-12-01}, journal = {Current Opinion in Cell Biology}, volume = {5}, pages = {966-70}, abstract = {In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases. |
Bystroff, C; Baker, D; Fletterick, R J; Agard, D A PRISM: application to the solution of two protein structures Journal Article Acta crystallographica. Section D, 49 , pp. 440-8, 1993, ISSN: 0907-4449. @article{326, title = {PRISM: application to the solution of two protein structures}, author = { C Bystroff and D Baker and R J Fletterick and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff93A.pdf}, issn = {0907-4449}, year = {1993}, date = {1993-09-01}, journal = {Acta crystallographica. Section D}, volume = {49}, pages = {440-8}, abstract = {The previous paper described a phase-refinement strategy for protein crystallography which exploited the information that proteins consist of connected linear chains of atoms. Here the method is applied to a molecular-replacement problem, the structure of the protease inhibitor ecotin bound to trypsin, and a single isomorphous replacement problem, the structure of the N-terminal domain of apolipoprotein E. The starting phases for the ecotin-trypsin complex were based on a partial model (trypsin) containing 61% of the atoms in the complex. Iterative skeletonization gave better results than either solvent flattening or twofold non-crystallographic symmetry averaging as measured by the reduction in the free R factor [Br"unger (1992). Nature (London), 355, 472-474]. Protection of the trypsin density during the course of the refinement greatly improved the performance of both skeletonizing and solvent flattening. In the case of apolipoprotein E, previous attempts using solvent flattening had failed to improve the SIR phases to the point of obtaining an interpretable map. The combination of iterative skeletonization and solvent flattening decreased the phase error with respect to the final refined structure, significantly more than solvent flattening alone. The final maps generated by the skeletonization procedure for both the ecotin-trypsin complex and apolipoprotein E were readily interpretable.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The previous paper described a phase-refinement strategy for protein crystallography which exploited the information that proteins consist of connected linear chains of atoms. Here the method is applied to a molecular-replacement problem, the structure of the protease inhibitor ecotin bound to trypsin, and a single isomorphous replacement problem, the structure of the N-terminal domain of apolipoprotein E. The starting phases for the ecotin-trypsin complex were based on a partial model (trypsin) containing 61% of the atoms in the complex. Iterative skeletonization gave better results than either solvent flattening or twofold non-crystallographic symmetry averaging as measured by the reduction in the free R factor [Br"unger (1992). Nature (London), 355, 472-474]. Protection of the trypsin density during the course of the refinement greatly improved the performance of both skeletonizing and solvent flattening. In the case of apolipoprotein E, previous attempts using solvent flattening had failed to improve the SIR phases to the point of obtaining an interpretable map. The combination of iterative skeletonization and solvent flattening decreased the phase error with respect to the final refined structure, significantly more than solvent flattening alone. The final maps generated by the skeletonization procedure for both the ecotin-trypsin complex and apolipoprotein E were readily interpretable. |
Baker, D; Bystroff, C; Fletterick, R J; Agard, D A PRISM: topologically constrained phased refinement for macromolecular crystallography Journal Article Acta crystallographica. Section D, 49 , pp. 429-39, 1993, ISSN: 0907-4449. @article{323, title = {PRISM: topologically constrained phased refinement for macromolecular crystallography}, author = { D Baker and C Bystroff and R J Fletterick and D A Agard}, issn = {0907-4449}, year = {1993}, date = {1993-09-01}, journal = {Acta crystallographica. Section D}, volume = {49}, pages = {429-39}, abstract = {We describe the further development of phase refinement by iterative skeletonization (PRISM), a recently introduced phase-refinement strategy [Wilson & Agard (1993). Acta Cryst. A49, 97-104] which makes use of the information that proteins consist of connected linear chains of atoms. An initial electron-density map is generated with inaccurate phases derived from a partial structure or from isomorphous replacement. A linear connected skeleton is then constructed from the map using a modified version of Greertextquoterights algorithm [Greer (1985). Methods Enzymol. 115, 206-226] and a new map is created from the skeleton. This textquoterightskeletonizedtextquoteright map is Fourier transformed to obtained new phases, which are combined with any starting-phase information and the experimental structure-factor amplitudes to produce a new map. The procedure is iterated until convergence is reached. In this paper significant improvements to the method are described as is a challenging molecular-replacement test case in which initial phases are calculated from a model containing only one third of the atoms of the intact protein. Application of the skeletonization procedure yields an easily interpretable map. In contrast, application of solvent flattening does not significantly improve the starting map. The iterative skeletonization procedure performs well in the presence of random noise and missing data, but requires Fourier data to at least 3.0 A. The constraints of linearity and connectedness prove strong enough to restore not only missing phase information, but also missing amplitudes. This enables the use of a powerful statistical test, analogous to the textquoterightfree R factortextquoteright of conventional refinement [Br"unger (1992). Nature (London), 355, 472-474], for optimizing the performance of the skeletonization procedure. In the accompanying paper, we describe the application of the method to the solution of the structure of the protease inhibitor ecotin bound to trypsin and to a single isomorphous replacement problem.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe the further development of phase refinement by iterative skeletonization (PRISM), a recently introduced phase-refinement strategy [Wilson & Agard (1993). Acta Cryst. A49, 97-104] which makes use of the information that proteins consist of connected linear chains of atoms. An initial electron-density map is generated with inaccurate phases derived from a partial structure or from isomorphous replacement. A linear connected skeleton is then constructed from the map using a modified version of Greertextquoterights algorithm [Greer (1985). Methods Enzymol. 115, 206-226] and a new map is created from the skeleton. This textquoterightskeletonizedtextquoteright map is Fourier transformed to obtained new phases, which are combined with any starting-phase information and the experimental structure-factor amplitudes to produce a new map. The procedure is iterated until convergence is reached. In this paper significant improvements to the method are described as is a challenging molecular-replacement test case in which initial phases are calculated from a model containing only one third of the atoms of the intact protein. Application of the skeletonization procedure yields an easily interpretable map. In contrast, application of solvent flattening does not significantly improve the starting map. The iterative skeletonization procedure performs well in the presence of random noise and missing data, but requires Fourier data to at least 3.0 A. The constraints of linearity and connectedness prove strong enough to restore not only missing phase information, but also missing amplitudes. This enables the use of a powerful statistical test, analogous to the textquoterightfree R factortextquoteright of conventional refinement [Br"unger (1992). Nature (London), 355, 472-474], for optimizing the performance of the skeletonization procedure. In the accompanying paper, we describe the application of the method to the solution of the structure of the protease inhibitor ecotin bound to trypsin and to a single isomorphous replacement problem. |
Ruohola-Baker, H; Grell, E; Chou, T B; Baker, D; Jan, L Y; Jan, Y N Spatially localized rhomboid is required for establishment of the dorsal-ventral axis in Drosophila oogenesis Journal Article Cell, 73 , pp. 953-65, 1993, ISSN: 0092-8674. @article{327, title = {Spatially localized rhomboid is required for establishment of the dorsal-ventral axis in Drosophila oogenesis}, author = { H Ruohola-Baker and E Grell and T B Chou and D Baker and L Y Jan and Y N Jan}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/ruohola-baker93A-1.pdf}, issn = {0092-8674}, year = {1993}, date = {1993-06-01}, journal = {Cell}, volume = {73}, pages = {953-65}, abstract = {The establishment of dorsal-ventral asymmetry of the Drosophila embryo requires a group of genes that act maternally. None of the previously identified dorsal-ventral axis genes are known to produce asymmetrically localized gene products during oogenesis. We show that rhomboid (rho), a novel member of this group, encodes a protein that is localized on the apical surface of the dorsal-anterior follicle cells surrounding the oocyte. Loss of rho function causes ventralization of the eggshell and the embryo, whereas ectopic expression leads to dorsalization of both structures. Thus, spatially restricted rho is necessary and sufficient for dorsal-ventral axis formation. We propose, based on these observations and double mutant experiments, that the spatially restricted rho protein leads to selective activation of the epidermal growth factor receptor in the dorsal follicle cells and subsequently the specification of the dorsal follicle cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The establishment of dorsal-ventral asymmetry of the Drosophila embryo requires a group of genes that act maternally. None of the previously identified dorsal-ventral axis genes are known to produce asymmetrically localized gene products during oogenesis. We show that rhomboid (rho), a novel member of this group, encodes a protein that is localized on the apical surface of the dorsal-anterior follicle cells surrounding the oocyte. Loss of rho function causes ventralization of the eggshell and the embryo, whereas ectopic expression leads to dorsalization of both structures. Thus, spatially restricted rho is necessary and sufficient for dorsal-ventral axis formation. We propose, based on these observations and double mutant experiments, that the spatially restricted rho protein leads to selective activation of the epidermal growth factor receptor in the dorsal follicle cells and subsequently the specification of the dorsal follicle cells. |
Baker, D; Krukowski, A E; Agard, D A Uniqueness and the ab initio phase problem in macromolecular crystallography Journal Article Acta crystallographica. Section D, 49 , pp. 186-92, 1993, ISSN: 0907-4449. @article{325, title = {Uniqueness and the ab initio phase problem in macromolecular crystallography}, author = { D Baker and A E Krukowski and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker93C.pdf}, issn = {0907-4449}, year = {1993}, date = {1993-01-01}, journal = {Acta crystallographica. Section D}, volume = {49}, pages = {186-92}, abstract = {The crystallographic phase problem is indeterminate in the absence of additional chemical information. A successful ab initio approach to the macromolecular phase problem must employ sufficient chemical constraints to limit the solutions to a manageably small number. Here we show that commonly employed chemical constraints - positivity, atomicity and a solvent boundary - leave the phase problem greatly underdetermined for Fourier data sets of moderate (2.5-3.0 A) resolution. Entropy maximization is also beset by multiple false solutions: electron-density maps are readily generated which satisfy the same Fourier amplitude constraints but have higher entropies than the true solution. We conclude that a successful ab initio approach must make use of high-resolution Fourier data and/or stronger chemical constraints. One such constraint is the connectivity of the macromolecule. We describe a rapid algorithm for measuring the connectivity of a map, and show its utility in reducing the multiplicity of solutions to the phase problem.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The crystallographic phase problem is indeterminate in the absence of additional chemical information. A successful ab initio approach to the macromolecular phase problem must employ sufficient chemical constraints to limit the solutions to a manageably small number. Here we show that commonly employed chemical constraints - positivity, atomicity and a solvent boundary - leave the phase problem greatly underdetermined for Fourier data sets of moderate (2.5-3.0 A) resolution. Entropy maximization is also beset by multiple false solutions: electron-density maps are readily generated which satisfy the same Fourier amplitude constraints but have higher entropies than the true solution. We conclude that a successful ab initio approach must make use of high-resolution Fourier data and/or stronger chemical constraints. One such constraint is the connectivity of the macromolecule. We describe a rapid algorithm for measuring the connectivity of a map, and show its utility in reducing the multiplicity of solutions to the phase problem. |
D, Baker; HS, Chan; KA, Dill Coordinate-Space Formulation of Polymer Lattice Cluster Theory Journal Article Journal of Chemical Physics, 1993. @article{324, title = {Coordinate-Space Formulation of Polymer Lattice Cluster Theory}, author = { Baker D and Chan HS and Dill KA}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker93B.pdf}, year = {1993}, date = {1993-01-01}, journal = {Journal of Chemical Physics}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
1992 |
Baker, D; Silen, J L; Agard, D A Protease pro region required for folding is a potent inhibitor of the mature enzyme Journal Article Proteins, 12 , pp. 339-44, 1992, ISSN: 0887-3585. @article{328, title = {Protease pro region required for folding is a potent inhibitor of the mature enzyme}, author = { D Baker and J L Silen and D A Agard}, issn = {0887-3585}, year = {1992}, date = {1992-04-01}, journal = {Proteins}, volume = {12}, pages = {339-44}, abstract = {alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.}, keywords = {}, pubstate = {published}, tppubtype = {article} } alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure. |
Baker, D; Sohl, J L; Agard, D A A protein-folding reaction under kinetic control Journal Article Nature, 356 , pp. 263-5, 1992, ISSN: 0028-0836. @article{329, title = {A protein-folding reaction under kinetic control}, author = { D Baker and J L Sohl and D A Agard}, issn = {0028-0836}, year = {1992}, date = {1992-03-01}, journal = {Nature}, volume = {356}, pages = {263-5}, abstract = {Synthesis of alpha-lytic protease is as a precursor containing a 166 amino-acid pro region transiently required for the correct folding of the protease domain. By omitting the pro region in an in vitro refolding reaction we trapped an inactive, but folding competent state (I) having an expanded radius yet native-like secondary structure. The I state is stable for weeks at physiological pH in the absence of denaturant, but rapidly folds to the active, native state on addition of the pro region as a separate polypeptide chain. The mechanism of action of the pro region is distinct from that of the chaperonins: rather than reducing the rate of off-pathway reactions, the pro region accelerates the rate-limiting step on the folding pathway by more than 10(7). Because both the I and native states are stable under identical conditions with no detectable interconversion, the folding of alpha-lytic protease must be under kinetic and not thermodynamic control.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Synthesis of alpha-lytic protease is as a precursor containing a 166 amino-acid pro region transiently required for the correct folding of the protease domain. By omitting the pro region in an in vitro refolding reaction we trapped an inactive, but folding competent state (I) having an expanded radius yet native-like secondary structure. The I state is stable for weeks at physiological pH in the absence of denaturant, but rapidly folds to the active, native state on addition of the pro region as a separate polypeptide chain. The mechanism of action of the pro region is distinct from that of the chaperonins: rather than reducing the rate of off-pathway reactions, the pro region accelerates the rate-limiting step on the folding pathway by more than 10(7). Because both the I and native states are stable under identical conditions with no detectable interconversion, the folding of alpha-lytic protease must be under kinetic and not thermodynamic control. |
1991 |
Ruohola, H; Bremer, K A; Baker, D; Swedlow, J R; Jan, L Y; Jan, Y N Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila. Journal Article Cell, 66 , pp. 433-49, 1991, ISSN: 0092-8674. @article{330, title = {Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila.}, author = { H Ruohola and K A Bremer and D Baker and J R Swedlow and L Y Jan and Y N Jan}, issn = {0092-8674}, year = {1991}, date = {1991-08-01}, journal = {Cell}, volume = {66}, pages = {433-49}, abstract = {Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte. |
1990 |
Baker, D; Wuestehube, L; Schekman, R; Botstein, D; Segev, N GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction Journal Article Proceedings of the National Academy of Sciences of the United States of America, 87 , pp. 355-9, 1990, ISSN: 0027-8424. @article{331, title = {GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction}, author = { D Baker and L Wuestehube and R Schekman and D Botstein and N Segev}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker90A.pdf}, issn = {0027-8424}, year = {1990}, date = {1990-01-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {87}, pages = {355-9}, abstract = {The 21-kDa GTP-binding Ypt1 protein (Ypt1p) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Ypt1 antibodies block transport; this inhibition is alleviated by competition with excess purified Ypt1p produced in bacteria. Furthermore, extracts of cells carrying the mutation ypt1-1 are defective in transport, but transport is restored if a cytosolic fraction from wild-type cells is provided. The in vitro transport reaction also requires physiological levels of Ca2+. However, Ypt1p functions independently of Ca2+. First, buffering the free Ca2+ at concentrations ranging from 1 nM to 10 microM does not relieve inhibition by Ypt1 antibodies. Second, consumption of a Ca2+-requiring intermediate that accumulates in Ca2+-deficient incubations is not inhibited by anti-Ypt1 antibodies, although completion of transport requires ATP and an N-ethylmaleimide-sensitive factor. Thus, Ypt1p and Ca2+ are required at distinct steps.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The 21-kDa GTP-binding Ypt1 protein (Ypt1p) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Ypt1 antibodies block transport; this inhibition is alleviated by competition with excess purified Ypt1p produced in bacteria. Furthermore, extracts of cells carrying the mutation ypt1-1 are defective in transport, but transport is restored if a cytosolic fraction from wild-type cells is provided. The in vitro transport reaction also requires physiological levels of Ca2+. However, Ypt1p functions independently of Ca2+. First, buffering the free Ca2+ at concentrations ranging from 1 nM to 10 microM does not relieve inhibition by Ypt1 antibodies. Second, consumption of a Ca2+-requiring intermediate that accumulates in Ca2+-deficient incubations is not inhibited by anti-Ypt1 antibodies, although completion of transport requires ATP and an N-ethylmaleimide-sensitive factor. Thus, Ypt1p and Ca2+ are required at distinct steps. |
1989 |
Baker, D; Schekman, R Reconstitution of protein transport using broken yeast spheroplasts Journal Article Methods in Cell Biology, 31 , pp. 127-41, 1989, ISSN: 0091-679X. @article{569, title = {Reconstitution of protein transport using broken yeast spheroplasts}, author = { D Baker and R Schekman}, issn = {0091-679X}, year = {1989}, date = {1989-00-01}, journal = {Methods in Cell Biology}, volume = {31}, pages = {127-41}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
1988 |
Baker, D; Hicke, L; Rexach, M; Schleyer, M; Schekman, R Reconstitution of SEC gene product-dependent intercompartmental protein transport Journal Article Cell, 54 , pp. 335-44, 1988, ISSN: 0092-8674. @article{332, title = {Reconstitution of SEC gene product-dependent intercompartmental protein transport}, author = { D Baker and L Hicke and M Rexach and M Schleyer and R Schekman}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker88.pdf}, issn = {0092-8674}, year = {1988}, date = {1988-07-01}, journal = {Cell}, volume = {54}, pages = {335-44}, abstract = {Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors. |
Payne, G S; Baker, D; van Tuinen, E; Schekman, R Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast Journal Article The Journal of cell biology, 106 , pp. 1453-61, 1988, ISSN: 0021-9525. @article{333, title = {Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast}, author = { G S Payne and D Baker and E van Tuinen and R Schekman}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/payne88A.pdf}, issn = {0021-9525}, year = {1988}, date = {1988-05-01}, journal = {The Journal of cell biology}, volume = {106}, pages = {1453-61}, abstract = {Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells. |