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1997 |
Scalley, M L; Yi, Q; Gu, H; McCormack, A; Yates, J R; Baker, David Kinetics of folding of the IgG binding domain of peptostreptococcal protein L. Journal Article Biochemistry, 36 , pp. 3373-82, 1997, ISSN: 0006-2960. @article{34, title = {Kinetics of folding of the IgG binding domain of peptostreptococcal protein L.}, author = { M L Scalley and Q Yi and H Gu and A McCormack and J R Yates and David Baker}, issn = {0006-2960}, year = {1997}, date = {1997-03-01}, journal = {Biochemistry}, volume = {36}, pages = {3373-82}, abstract = {The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar. (3) Mass spectra from pulsed HD exchange refolding experiments showed that a species with very little protection from exchange is converted to a fully protected species (the native state) at a rate very similar to that of the overall change in tryptophan fluorescence; no intervening partially protected species were observed. (4) Rate constants (in H2O) and m values for folding and unfolding determined by fitting observed relaxation rates obtained over a broad range of denaturant concentrations to a two-state model were consistent with the equilibrium parameters deltaG and m: -RT ln(k(u)/k(f))/deltaG(U)H2O = 1.02; (m(u) + m(f))/m = 1.08. In contrast to results with a number of other proteins, there was no deviation from linearity in plots of ln k(obs) versus guanidine at low guanidine concentrations, both in the presence and absence of 0.4 M Na2SO4, suggesting that significantly stabilized intermediates do not accumulate during folding. Although all of the change in fluorescence signal during folding in phosphate buffer was accounted for by the simple exponential describing the overall folding reaction, fluorescence-quenching experiments using sodium iodide revealed a small reduction in the extent of quenching of the protein within the first two milliseconds after initiation of refolding in low concentrations of guanidine, suggesting a partial collapse of the unfolded chain may occur under these conditions. Comparison with results on the structurally and functionally similar IgG binding domain of streptococcal protein G show intriguing differences in the folding of the two proteins.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar. (3) Mass spectra from pulsed HD exchange refolding experiments showed that a species with very little protection from exchange is converted to a fully protected species (the native state) at a rate very similar to that of the overall change in tryptophan fluorescence; no intervening partially protected species were observed. (4) Rate constants (in H2O) and m values for folding and unfolding determined by fitting observed relaxation rates obtained over a broad range of denaturant concentrations to a two-state model were consistent with the equilibrium parameters deltaG and m: -RT ln(k(u)/k(f))/deltaG(U)H2O = 1.02; (m(u) + m(f))/m = 1.08. In contrast to results with a number of other proteins, there was no deviation from linearity in plots of ln k(obs) versus guanidine at low guanidine concentrations, both in the presence and absence of 0.4 M Na2SO4, suggesting that significantly stabilized intermediates do not accumulate during folding. Although all of the change in fluorescence signal during folding in phosphate buffer was accounted for by the simple exponential describing the overall folding reaction, fluorescence-quenching experiments using sodium iodide revealed a small reduction in the extent of quenching of the protein within the first two milliseconds after initiation of refolding in low concentrations of guanidine, suggesting a partial collapse of the unfolded chain may occur under these conditions. Comparison with results on the structurally and functionally similar IgG binding domain of streptococcal protein G show intriguing differences in the folding of the two proteins. |
Rank, J A; Baker, David A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding Journal Article Protein science, 6 , pp. 347-54, 1997, ISSN: 0961-8368. @article{35, title = {A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding}, author = { J A Rank and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/rank97A.pdf}, issn = {0961-8368}, year = {1997}, date = {1997-02-01}, journal = {Protein science}, volume = {6}, pages = {347-54}, abstract = {To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.}, keywords = {}, pubstate = {published}, tppubtype = {article} } To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error. |
Bystroff, C; Baker, David Blind predictions of local protein structure in CASP2 targets using the I-sites library Journal Article Proteins, Suppl 1 , pp. 167-71, 1997, ISSN: 0887-3585. @article{30, title = {Blind predictions of local protein structure in CASP2 targets using the I-sites library}, author = { C Bystroff and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff97A.pdf}, issn = {0887-3585}, year = {1997}, date = {1997-00-01}, journal = {Proteins}, volume = {Suppl 1}, pages = {167-71}, abstract = {Blind predictions of the local structure of nine CASP2 targets were made using the I-sites library of short sequence--structure motifs, revealing strengths and weaknesses in this new knowledge-based method. Many turns between secondary structural elements were accurately predicted. Estimates of the confidence of prediction correlated well with the accuracy over the whole set. Bias toward structures used to develop the library was minimal, probably because of the extensive use of cross-validation. However, helix positions were better predicted by the PHD program. The method is likely to be sensitive to the quality of the sequence alignment. A general measure for evaluating local structure predictions is suggested.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Blind predictions of the local structure of nine CASP2 targets were made using the I-sites library of short sequence--structure motifs, revealing strengths and weaknesses in this new knowledge-based method. Many turns between secondary structural elements were accurately predicted. Estimates of the confidence of prediction correlated well with the accuracy over the whole set. Bias toward structures used to develop the library was minimal, probably because of the extensive use of cross-validation. However, helix positions were better predicted by the PHD program. The method is likely to be sensitive to the quality of the sequence alignment. A general measure for evaluating local structure predictions is suggested. |
Yi, Q; Scalley, M L; Simons, K T; Gladwin, S T; Baker, David Characterization of the free energy spectrum of peptostreptococcal protein L Journal Article Folding & design, 2 , pp. 271-80, 1997, ISSN: 1359-0278. @article{27, title = {Characterization of the free energy spectrum of peptostreptococcal protein L}, author = { Q Yi and M L Scalley and K T Simons and S T Gladwin and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/yi97A.pdf}, issn = {1359-0278}, year = {1997}, date = {1997-00-01}, journal = {Folding & design}, volume = {2}, pages = {271-80}, abstract = {BACKGROUND: Native state hydrogen/deuterium exchange studies on cytochrome c and RNase H revealed the presence of excited states with partially formed native structure. We set out to determine whether such excited states are populated for a very small and simple protein, the IgG-binding domain of peptostreptococcal protein L. RESULTS: Hydrogen/deuterium exchange data on protein L in 0-1.2 M guanidine fit well to a simple model in which the only contributions to exchange are denaturant-independent local fluctuations and global unfolding. A substantial discrepancy emerged between unfolding free energy estimates from hydrogen/deuterium exchange and linear extrapolation of earlier guanidine denaturation experiments. A better determined estimate of the free energy of unfolding obtained by global analysis of a series of thermal denaturation experiments in the presence of 0-3 M guanidine was in good agreement with the estimate from hydrogen/deuterium exchange. CONCLUSIONS: For protein L under native conditions, there do not appear to be partially folded states with free energies intermediate between that of the folded and unfolded states. The linear extrapolation method significantly underestimates the free energy of folding of protein L due to deviations from linearity in the dependence of the free energy on the denaturant concentration.}, keywords = {}, pubstate = {published}, tppubtype = {article} } BACKGROUND: Native state hydrogen/deuterium exchange studies on cytochrome c and RNase H revealed the presence of excited states with partially formed native structure. We set out to determine whether such excited states are populated for a very small and simple protein, the IgG-binding domain of peptostreptococcal protein L. RESULTS: Hydrogen/deuterium exchange data on protein L in 0-1.2 M guanidine fit well to a simple model in which the only contributions to exchange are denaturant-independent local fluctuations and global unfolding. A substantial discrepancy emerged between unfolding free energy estimates from hydrogen/deuterium exchange and linear extrapolation of earlier guanidine denaturation experiments. A better determined estimate of the free energy of unfolding obtained by global analysis of a series of thermal denaturation experiments in the presence of 0-3 M guanidine was in good agreement with the estimate from hydrogen/deuterium exchange. CONCLUSIONS: For protein L under native conditions, there do not appear to be partially folded states with free energies intermediate between that of the folded and unfolded states. The linear extrapolation method significantly underestimates the free energy of folding of protein L due to deviations from linearity in the dependence of the free energy on the denaturant concentration. |
1996 |
Bystroff, C; Simons, K T; Han, K F; Baker, David Local sequence-structure correlations in proteins Journal Article Current opinion in biotechnology, 7 , pp. 417-21, 1996, ISSN: 0958-1669. @article{214, title = {Local sequence-structure correlations in proteins}, author = { C Bystroff and K T Simons and K F Han and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff96A.pdf}, issn = {0958-1669}, year = {1996}, date = {1996-08-01}, journal = {Current opinion in biotechnology}, volume = {7}, pages = {417-21}, abstract = {Considerable progress has been made in understanding the relationship between local amino acid sequence and local protein structure. Recent highlights include numerous studies of the structures adopted by short peptides, new approaches to correlating sequence patterns with structure patterns, and folding simulations using simple potentials.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Considerable progress has been made in understanding the relationship between local amino acid sequence and local protein structure. Recent highlights include numerous studies of the structures adopted by short peptides, new approaches to correlating sequence patterns with structure patterns, and folding simulations using simple potentials. |
Han, K F; Baker, David Global properties of the mapping between local amino acid sequence and local structure in proteins Journal Article Proceedings of the National Academy of Sciences of the United States of America, 93 , pp. 5814-8, 1996, ISSN: 0027-8424. @article{215, title = {Global properties of the mapping between local amino acid sequence and local structure in proteins}, author = { K F Han and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/han96A.pdf}, issn = {0027-8424}, year = {1996}, date = {1996-06-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {93}, pages = {5814-8}, abstract = {Local protein structure prediction efforts have consistently failed to exceed approximately 70% accuracy. We characterize the degeneracy of the mapping from local sequence to local structure responsible for this failure by investigating the extent to which similar sequence segments found in different proteins adopt similar three-dimensional structures. Sequence segments 3-15 residues in length from 154 different protein families are partitioned into neighborhoods containing segments with similar sequences using cluster analysis. The consistency of the sequence-to-structure mapping is assessed by comparing the local structures adopted by sequence segments in the same neighborhood in proteins of known structure. In the 154 families, 45% and 28% of the positions occur in neighborhoods in which one and two local structures predominate, respectively. The sequence patterns that characterize the neighborhoods in the first class probably include virtually all of the short sequence motifs in proteins that consistently occur in a particular local structure. These patterns, many of which occur in transitions between secondary structural elements, are an interesting combination of previously studied and novel motifs. The identification of sequence patterns that consistently occur in one or a small number of local structures in proteins should contribute to the prediction of protein structure from sequence.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Local protein structure prediction efforts have consistently failed to exceed approximately 70% accuracy. We characterize the degeneracy of the mapping from local sequence to local structure responsible for this failure by investigating the extent to which similar sequence segments found in different proteins adopt similar three-dimensional structures. Sequence segments 3-15 residues in length from 154 different protein families are partitioned into neighborhoods containing segments with similar sequences using cluster analysis. The consistency of the sequence-to-structure mapping is assessed by comparing the local structures adopted by sequence segments in the same neighborhood in proteins of known structure. In the 154 families, 45% and 28% of the positions occur in neighborhoods in which one and two local structures predominate, respectively. The sequence patterns that characterize the neighborhoods in the first class probably include virtually all of the short sequence motifs in proteins that consistently occur in a particular local structure. These patterns, many of which occur in transitions between secondary structural elements, are an interesting combination of previously studied and novel motifs. The identification of sequence patterns that consistently occur in one or a small number of local structures in proteins should contribute to the prediction of protein structure from sequence. |
Yi, Q; Baker, David Direct evidence for a two-state protein unfolding transition from hydrogen-deuterium exchange, mass spectrometry, and NMR Journal Article Protein science, 5 , pp. 1060-6, 1996, ISSN: 0961-8368. @article{24, title = {Direct evidence for a two-state protein unfolding transition from hydrogen-deuterium exchange, mass spectrometry, and NMR}, author = { Q Yi and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/yi96A.pdf}, issn = {0961-8368}, year = {1996}, date = {1996-06-01}, journal = {Protein science}, volume = {5}, pages = {1060-6}, abstract = {We use mass spectrometry in conjunction with hydrogen-deuterium exchange and NMR to characterize the conformational dynamics of the 62-residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (> 10(5)/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at approximately 0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR-derived estimates of the proton exchange rates.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We use mass spectrometry in conjunction with hydrogen-deuterium exchange and NMR to characterize the conformational dynamics of the 62-residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (> 10(5)/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at approximately 0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR-derived estimates of the proton exchange rates. |
1995 |
Han, K F; Baker, David Recurring local sequence motifs in proteins Journal Article Journal of molecular biology, 251 , pp. 176-87, 1995, ISSN: 0022-2836. @article{22, title = {Recurring local sequence motifs in proteins}, author = { K F Han and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/han95A.pdf}, issn = {0022-2836}, year = {1995}, date = {1995-08-01}, journal = {Journal of molecular biology}, volume = {251}, pages = {176-87}, abstract = {We describe a completely automated approach to identifying local sequence motifs that transcend protein family boundaries. Cluster analysis is used to identify recurring patterns of variation at single positions and in short segments of contiguous positions in multiple sequence alignments for a non-redundant set of protein families. Parallel experiments on simulated data sets constructed with the overall residue frequencies of proteins but not the inter-residue correlations show that naturally occurring protein sequences are significantly more clustered than the corresponding random sequences for window lengths ranging from one to 13 contiguous positions. The patterns of variation at single positions are not in general surprising: chemically similar amino acids tend to be grouped together. More interesting patterns emerge as the window length increases. The patterns of variation for longer window lengths are in part recognizable patterns of hydrophobic and hydrophilic residues, and in part less obvious combinations. A particularly interesting class of patterns features highly conserved glycine residues. The patterns provide a means to abstract the information contained in multiple sequence alignments and may be useful for comparison of distantly related sequences or sequence families and for protein structure prediction.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a completely automated approach to identifying local sequence motifs that transcend protein family boundaries. Cluster analysis is used to identify recurring patterns of variation at single positions and in short segments of contiguous positions in multiple sequence alignments for a non-redundant set of protein families. Parallel experiments on simulated data sets constructed with the overall residue frequencies of proteins but not the inter-residue correlations show that naturally occurring protein sequences are significantly more clustered than the corresponding random sequences for window lengths ranging from one to 13 contiguous positions. The patterns of variation at single positions are not in general surprising: chemically similar amino acids tend to be grouped together. More interesting patterns emerge as the window length increases. The patterns of variation for longer window lengths are in part recognizable patterns of hydrophobic and hydrophilic residues, and in part less obvious combinations. A particularly interesting class of patterns features highly conserved glycine residues. The patterns provide a means to abstract the information contained in multiple sequence alignments and may be useful for comparison of distantly related sequences or sequence families and for protein structure prediction. |
Gu, H; Yi, Q; Bray, S T; Riddle, D S; Shiau, A K; Baker, David A phage display system for studying the sequence determinants of protein folding Journal Article Protein science, 4 , pp. 1108-17, 1995, ISSN: 0961-8368. @article{216, title = {A phage display system for studying the sequence determinants of protein folding}, author = { H Gu and Q Yi and S T Bray and D S Riddle and A K Shiau and David Baker}, issn = {0961-8368}, year = {1995}, date = {1995-06-01}, journal = {Protein science}, volume = {4}, pages = {1108-17}, abstract = {We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein. |
1994 |
Baker, D; Agard, D A Influenza hemagglutinin: kinetic control of protein function. Journal Article Structure (London, England : 1993), 2 , pp. 907-10, 1994, ISSN: 0969-2126. @article{572, title = {Influenza hemagglutinin: kinetic control of protein function.}, author = { D Baker and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/influenzahemagglutinin_Baker1994.pdf}, issn = {0969-2126}, year = {1994}, date = {1994-10-01}, journal = {Structure (London, England : 1993)}, volume = {2}, pages = {907-10}, abstract = {In response to decreased pH, influenza hemagglutinin changes to a more stable conformation. Such changes, which can be controlled thermodynamically or kinetically, are the method by which many biological textquoterightswitchestextquoteright are thrown.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In response to decreased pH, influenza hemagglutinin changes to a more stable conformation. Such changes, which can be controlled thermodynamically or kinetically, are the method by which many biological textquoterightswitchestextquoteright are thrown. |
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. |