Amie McClellan | Bennington College (original) (raw)
Papers by Amie McClellan
Molecular Cell, Oct 1, 2006
The ring-shaped hetero-oligomeric chaperonin TRiC/ CCT uses ATP to fold a diverse subset of eukar... more The ring-shaped hetero-oligomeric chaperonin TRiC/ CCT uses ATP to fold a diverse subset of eukaryotic proteins. To define the basis of TRiC/CCT substrate recognition, we mapped the chaperonin interactions with the VHL tumor suppressor. VHL has two well-defined TRiC binding determinants. Each determinant contacts a specific subset of chaperonin subunits, indicating that TRiC paralogs exhibit distinct but overlapping specificities. The substrate binding site in these subunits localizes to a helical region in the apical domains that is structurally equivalent to that of bacterial chaperonins. Transferring the distal portion of helix 11 between TRiC subunits suffices to transfer specificity for a given substrate motif. We conclude that the architecture of the substrate binding domain is evolutionarily conserved among eukaryotic and bacterial chaperonins. The unique combination of specificity and plasticity in TRiC substrate binding may diversify the range of motifs recognized by this chaperonin and contribute to its unique ability to fold eukaryotic proteins.
Genetics, Oct 1, 2000
The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action... more The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote posttranslational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wildtype cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.
Journal of Microbiology & Biology Education
The Genomics Education Partnership (GEP) engages students in a course-based undergraduate researc... more The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit.
This article cites 45 articles, 16 of which can be accessed free
Molecular and Cellular Biology, 2003
The degree of cooperation and redundancy between different chaperones is an important problem in ... more The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell. Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex. VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells. Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells. Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70. VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4 , a sub...
Cell, 2005
In eukaryotes, in vitro and Judith Frydman* experiments demonstrate a requirement for Hsp70 in De... more In eukaryotes, in vitro and Judith Frydman* experiments demonstrate a requirement for Hsp70 in Department of Biological Sciences and BioX Program the degradation of some misfolded proteins (Bercovich Stanford University et al., 1997), while in vivo experiments in yeast implicate Stanford, California 94305 the Hsp70 cofactor Ydj1p in this process (Lee et al., 1996). However, these studies do not clarify the question of how chaperones contribute to the triage deci-Summary sion that targets polypeptides for degradation. Understanding how chaperones help select nonnative proteins The mechanisms by which molecular chaperones asfor degradation has been hindered by the use of grossly sist quality control of cytosolic proteins are poorly misfolded or "abnormal" polypeptides as models. Given understood. Analysis of the chaperone requirements that these proteins cannot fold under any circumstance for degradation of misfolded variants of a cytosolic and can only either be degraded or aggregate, they are protein, the VHL tumor suppressor, reveals that disnot adequate to address the mechanism of a physiotinct chaperone pathways mediate its folding and logical triage decision. Based on these studies, a simquality control. While both folding and degradation of ple model for protein quality control was proposed, VHL require Hsp70, the chaperonin TRiC is essential whereby chaperones perform a passive "holding" funcfor folding but is dispensable for degradation. Contion, maintaining the solubility of misfolded intermediversely, the chaperone Hsp90 neither participates in ates, thus facilitating sampling by the ubiquitination VHL folding nor is required to maintain misfolded machinery (Wickner et al., 1999). In this view, the role VHL solubility but is essential for its degradation. The of chaperones in quality control is an extension of their cochaperone HOP/Sti1p also participates in VHL qualprimary role in folding, in which repeated attempts to ity control and may direct the triage decision by fold a defective protein, through cycles of chaperone bridging the Hsp70-Hsp90 interaction. Our finding binding and release to the bulk solution, would eventuthat a distinct chaperone complex is uniquely really result in passive partitioning to the degradation maquired for quality control provides evidence for active chinery (Wickner et al., 1999). A distinguishing feature and specific chaperone participation in triage deciof this model is that the requirement for chaperones in sions and suggests that a hierarchy of chaperone inquality control stems primarily from the need to mainteractions can control the alternate fates of a cytotain the solubility of misfolded intermediates and is solic protein. thus nonspecific. Consequently, any chaperone system capable of binding a nonnative polypeptide may assist
Achieving the correct balance between folding and degradation of misfolded proteins is critical f... more Achieving the correct balance between folding and degradation of misfolded proteins is critical for cell viability. The importance of defining the mechanisms and factors that mediate cytoplasmic quality control is underscored by the growing list of diseases associated with protein misfolding and aggregation. Molecular chaperones assist protein folding and also facilitate degradation of misfolded polypeptides by the ubiquitin-proteasome system. Here we discuss emerging links between folding and degradation machineries and highlight challenges for future research.
Molecular chaperones have long been heralded as machines for fold-ing and salvaging proteins. How... more Molecular chaperones have long been heralded as machines for fold-ing and salvaging proteins. However, not every attempt to fold orrefold a protein can be successful. Chaperones are known to partici-pate in the degradation of misfolded polypeptides, but a direct linkbetween folding and degradation pathways has remained elusive. Tworecent reports show that the co-chaperone CHIP mediates ubiquitin-dependent degradation of substrates bound to heat-shock protein 70(Hsp70) and/or Hsp90.
Journal of Student Research
Sodium Dodecyl Sulfate, or SDS, is an anionic detergent with widespread use in industrial and hou... more Sodium Dodecyl Sulfate, or SDS, is an anionic detergent with widespread use in industrial and household cleaning products, scientific laboratories, and personal care products such as toothpaste and shampoo. The potential toxicity of SDS has been well-characterized in whole organism studies and its potential effects on the environment continue to be studied. Herein, we undertake a chemical-genetic screen to explore whether low concentrations of SDS have any discernible effects at the cellular level. Our screen of the homozygous diploid yeast deletion collection identified numerous gene deletions that confer sensitivity to SDS. Subsequent bioinformatic and biological analyses reveal that yeast unable to synthesize tryptophan are especially sensitive to the presence of SDS. Interestingly, even wild-type yeast with an intact tryptophan biosynthetic pathway exhibit growth defects in the presence of SDS on media lacking tryptophan. Altogether, we have shown that low levels of SDS, ...
Journal of Microbiology & Biology Education
Open Biology
Protein ubiquitination is of great cellular importance through its central role in processes such... more Protein ubiquitination is of great cellular importance through its central role in processes such as degradation, DNA repair, endocytosis and inflammation. Canonical ubiquitination takes place on lysine residues, but in the past 15 years non-lysine ubiquitination on serine, threonine and cysteine has been firmly established. With the emerging importance of non-lysine ubiquitination, it is crucial to identify the responsible molecular machinery and understand the mechanistic basis for non-lysine ubiquitination. Here, we first provide an overview of the literature that has documented non-lysine ubiquitination. Informed by these examples, we then discuss the molecular mechanisms and cellular implications of non-lysine ubiquitination, and conclude by outlining open questions and future perspectives in the field.
Cell Stress and Chaperones
Molecular and Cellular Biology
Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute ... more Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of larg...
Genetics, 2000
The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action... more The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an un...
Molecular Biology of the Cell, 1998
The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in... more The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant...
Molecular Cell, Oct 1, 2006
The ring-shaped hetero-oligomeric chaperonin TRiC/ CCT uses ATP to fold a diverse subset of eukar... more The ring-shaped hetero-oligomeric chaperonin TRiC/ CCT uses ATP to fold a diverse subset of eukaryotic proteins. To define the basis of TRiC/CCT substrate recognition, we mapped the chaperonin interactions with the VHL tumor suppressor. VHL has two well-defined TRiC binding determinants. Each determinant contacts a specific subset of chaperonin subunits, indicating that TRiC paralogs exhibit distinct but overlapping specificities. The substrate binding site in these subunits localizes to a helical region in the apical domains that is structurally equivalent to that of bacterial chaperonins. Transferring the distal portion of helix 11 between TRiC subunits suffices to transfer specificity for a given substrate motif. We conclude that the architecture of the substrate binding domain is evolutionarily conserved among eukaryotic and bacterial chaperonins. The unique combination of specificity and plasticity in TRiC substrate binding may diversify the range of motifs recognized by this chaperonin and contribute to its unique ability to fold eukaryotic proteins.
Genetics, Oct 1, 2000
The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action... more The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote posttranslational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wildtype cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.
Journal of Microbiology & Biology Education
The Genomics Education Partnership (GEP) engages students in a course-based undergraduate researc... more The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit.
This article cites 45 articles, 16 of which can be accessed free
Molecular and Cellular Biology, 2003
The degree of cooperation and redundancy between different chaperones is an important problem in ... more The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell. Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex. VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells. Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells. Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70. VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4 , a sub...
Cell, 2005
In eukaryotes, in vitro and Judith Frydman* experiments demonstrate a requirement for Hsp70 in De... more In eukaryotes, in vitro and Judith Frydman* experiments demonstrate a requirement for Hsp70 in Department of Biological Sciences and BioX Program the degradation of some misfolded proteins (Bercovich Stanford University et al., 1997), while in vivo experiments in yeast implicate Stanford, California 94305 the Hsp70 cofactor Ydj1p in this process (Lee et al., 1996). However, these studies do not clarify the question of how chaperones contribute to the triage deci-Summary sion that targets polypeptides for degradation. Understanding how chaperones help select nonnative proteins The mechanisms by which molecular chaperones asfor degradation has been hindered by the use of grossly sist quality control of cytosolic proteins are poorly misfolded or "abnormal" polypeptides as models. Given understood. Analysis of the chaperone requirements that these proteins cannot fold under any circumstance for degradation of misfolded variants of a cytosolic and can only either be degraded or aggregate, they are protein, the VHL tumor suppressor, reveals that disnot adequate to address the mechanism of a physiotinct chaperone pathways mediate its folding and logical triage decision. Based on these studies, a simquality control. While both folding and degradation of ple model for protein quality control was proposed, VHL require Hsp70, the chaperonin TRiC is essential whereby chaperones perform a passive "holding" funcfor folding but is dispensable for degradation. Contion, maintaining the solubility of misfolded intermediversely, the chaperone Hsp90 neither participates in ates, thus facilitating sampling by the ubiquitination VHL folding nor is required to maintain misfolded machinery (Wickner et al., 1999). In this view, the role VHL solubility but is essential for its degradation. The of chaperones in quality control is an extension of their cochaperone HOP/Sti1p also participates in VHL qualprimary role in folding, in which repeated attempts to ity control and may direct the triage decision by fold a defective protein, through cycles of chaperone bridging the Hsp70-Hsp90 interaction. Our finding binding and release to the bulk solution, would eventuthat a distinct chaperone complex is uniquely really result in passive partitioning to the degradation maquired for quality control provides evidence for active chinery (Wickner et al., 1999). A distinguishing feature and specific chaperone participation in triage deciof this model is that the requirement for chaperones in sions and suggests that a hierarchy of chaperone inquality control stems primarily from the need to mainteractions can control the alternate fates of a cytotain the solubility of misfolded intermediates and is solic protein. thus nonspecific. Consequently, any chaperone system capable of binding a nonnative polypeptide may assist
Achieving the correct balance between folding and degradation of misfolded proteins is critical f... more Achieving the correct balance between folding and degradation of misfolded proteins is critical for cell viability. The importance of defining the mechanisms and factors that mediate cytoplasmic quality control is underscored by the growing list of diseases associated with protein misfolding and aggregation. Molecular chaperones assist protein folding and also facilitate degradation of misfolded polypeptides by the ubiquitin-proteasome system. Here we discuss emerging links between folding and degradation machineries and highlight challenges for future research.
Molecular chaperones have long been heralded as machines for fold-ing and salvaging proteins. How... more Molecular chaperones have long been heralded as machines for fold-ing and salvaging proteins. However, not every attempt to fold orrefold a protein can be successful. Chaperones are known to partici-pate in the degradation of misfolded polypeptides, but a direct linkbetween folding and degradation pathways has remained elusive. Tworecent reports show that the co-chaperone CHIP mediates ubiquitin-dependent degradation of substrates bound to heat-shock protein 70(Hsp70) and/or Hsp90.
Journal of Student Research
Sodium Dodecyl Sulfate, or SDS, is an anionic detergent with widespread use in industrial and hou... more Sodium Dodecyl Sulfate, or SDS, is an anionic detergent with widespread use in industrial and household cleaning products, scientific laboratories, and personal care products such as toothpaste and shampoo. The potential toxicity of SDS has been well-characterized in whole organism studies and its potential effects on the environment continue to be studied. Herein, we undertake a chemical-genetic screen to explore whether low concentrations of SDS have any discernible effects at the cellular level. Our screen of the homozygous diploid yeast deletion collection identified numerous gene deletions that confer sensitivity to SDS. Subsequent bioinformatic and biological analyses reveal that yeast unable to synthesize tryptophan are especially sensitive to the presence of SDS. Interestingly, even wild-type yeast with an intact tryptophan biosynthetic pathway exhibit growth defects in the presence of SDS on media lacking tryptophan. Altogether, we have shown that low levels of SDS, ...
Journal of Microbiology & Biology Education
Open Biology
Protein ubiquitination is of great cellular importance through its central role in processes such... more Protein ubiquitination is of great cellular importance through its central role in processes such as degradation, DNA repair, endocytosis and inflammation. Canonical ubiquitination takes place on lysine residues, but in the past 15 years non-lysine ubiquitination on serine, threonine and cysteine has been firmly established. With the emerging importance of non-lysine ubiquitination, it is crucial to identify the responsible molecular machinery and understand the mechanistic basis for non-lysine ubiquitination. Here, we first provide an overview of the literature that has documented non-lysine ubiquitination. Informed by these examples, we then discuss the molecular mechanisms and cellular implications of non-lysine ubiquitination, and conclude by outlining open questions and future perspectives in the field.
Cell Stress and Chaperones
Molecular and Cellular Biology
Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute ... more Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of larg...
Genetics, 2000
The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action... more The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an un...
Molecular Biology of the Cell, 1998
The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in... more The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant...