Progress 10/01/04 to 09/30/09
Outputs
OUTPUTS: James Taylor and Stephen Smith, a postdoc and graduate student in my lab, respectively, have made mutations in an accessory protein, RcdA, and determined the effects of these mutations on CtrA proteolysis and protein localization. Jeremy Wilbur, a collaborating graduate student from the Fletterick and Brodsky labs at UCSF, solved the crystal structure of RcdA, which facilitated our structure-guided mutagenesis. Stephen Smith is reconstituting CtrA proteolysis in vitro using known and recently identified proteins that are required for this reaction in vivo. Our main question now is why the protease ClpXP can degrade CtrA in vitro, yet at least three accessory proteins are needed for the reaction in living Caulobacter crescentus. Two separate signals in CtrA are needed to specifiy degradation at the proper time in the cell cycle. One signal, in the receiver domain, is uncharacterized. An undergraduate, Aron Kamajaya, has mutated CtrA surface residues to identify amino acids necessary for CtrA proteolysis. He has analyzed the effects of these mutations on the CtrA half-life and its location in the cell. Aron and Stephen are preparing for immunoprecipitation experiments to identify proteins that bind to CtrA in Caulobacter. This step is important because, although the protease binds to the C-terminus of CtrA, no proteins have been identified that bind to the receiver domain, and none of the accessory proteins interact directly with CtrA. We hypothesize that there is at least one additional protein that recognizes the receiver domain degradation signal in CtrA. I have mentored Stephen and Aron in 2009, and Stephen has directed Aron's day-to-day activities. Stephen and I attended the 3rd International Caulobacter Meeting in June, 2009, where he presented a poster on his work. Stephen also attended the ASM general meeting in 2009. PARTICIPANTS: Individuals: Kathleen Ryan, PI, designed, performed, and interpreted experiments, co-wrote the manuscript for JMB, and mentored James Taylor, Stephen Smith, and Aron Kamajaya. James Taylor, a former postdoctoral researcher, designed, performed, and interpreted experiments and co-wrote the JMB manuscript. James is currently a postdoc in the Marczynski lab at McGill University. Stephen Smith, a current graduate student, designed, performed, and interpreted experiments, edited the JMB manuscript and mentored Aron Kamajaya. Aron Kamajaya, an undergraduate student, performed honors research in my lab. He designed, performed, and interpreted experiments on CtrA receiver domain mutants. He has given two talks at honors research symposia at UCB and will be attending graduate school at CalTech next year. Jeremy Wilbur, a collaborating graduate student, crystallized RcdA and solved its X-ray structure. He then advised us on mutageninzing surface residues of RcdA and co-wrote the JMB manuscript. Jeremy is now a postdoctoral researcher in the Heald lab at UC Berkeley. Training and professional development: This project has provided training and professional development for one former technician (Ben Sawicki), one postdoc, two graduate students, and one undergraduate. TARGET AUDIENCES: The target audience of this project is the scientific community seeking to understand cellular regulatory mechanisms that rely on controlled degradation of key proteins. Our efforts to disseminate knowledge included publication in scientific journals, presenting posters at meetings, and delivering seminars at other universities. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Changes in Knowledge: Our RcdA mutagenesis project revealed features of that accessory protein that are involved in CtrA degradation and subcellular localization of RcdA and CtrA. To our surprise, some mutations decoupled protein localization from rapid CtrA degradation. Before, it had been assumed that RcdA worked as an adaptor to bring CtrA to the protease at the cell pole, but our results indicate that RcdA plays an additional biochemical role. Our mutagenesis of CtrA has identified residues in the first alpha helix that specify cell cycle-regulated proteolysis. Changing these residues does not disrupt the normal functions of CtrA, only its degradation. This suggests that one or more proteins will recognize helix 1 as a step in degrading CtrA.
Publications
- Journal Articles: Taylor, J.A., Wilbur, J.D., Smith, S.C., and Ryan, K.R. (2009). Mutations that alter RcdA surface residues decouple protein localization and CtrA proteolysis in Caulobacter crescentus. J. Mol. Biol. 394: 46-60.
- Abstracts: Smith, S.C., Taylor, J.A., Wilbur, J.D., and Ryan, K.R. (2009). RcdA structure and function in regulated CtrA proteolysis. Abstract for Poster Presentation, 3rd International Caulobacter Meeting, Boston, MA.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: We completed a mutational analysis of conserved surface residues in RcdA to identify amino acids necessary for timed CtrA proteolysis during the cell cycle. We assayed CtrA degradation and protein localization in each strain containing a different rcdA mutation. We used a bioinformatic approach to predict a new set of amino acids in CtrA that specify when it is degraded during the cell cycle. We have constructed and tested three mutants for proper degradation so far. The RcdA experiments were performed by Stephen Smith, a graduate student who joined the lab in the spring of 2008. The CtrA experiments were performed by Aron Kamajaya, a new undergraduate researcher who joined the lab in November, 2008. I have trained both of these new students in our standard lab techniques, including cell cycle synchronization, fluorescence microscopy, pulse-chase assays, and flow cytometry. I have advised both Stephen and Aron on writing fellowship applications to secure outside funding, and Stephen is writing a new draft of our manuscript on RcdA to take into account his new findings. I have presented our results on RcdA in seminars at the University of Chicago, the University of Illinois at Chicago, the University of Iowa, and the Gordon Conference Signal Transduction in Microorganisms. PARTICIPANTS: James A. Taylor was a postdoctoral researcher who began our studies of RcdA structure and function. He left the lab in July, 2008, and now works as a postdoctoral researcher at McGill University. Stephen C. Smith is the graduate student who took over the RcdA project from James Taylor. Aron Kamajaya is an undergraduate who is studying CtrA proteolysis. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Prior to this year, it was thought that RcdA had to be positioned at the cell pole, along with CtrA, to promote CtrA degradation. Our work changing specific surface residues of RcdA uncovered mutants which are not located at the cell pole, but nevertheless are able to support CtrA proteolysis. This is a surprising result that has forced us to consider alternative models for the molecular function of RcdA.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Our lab and others have found that regulated CtrA proteolysis in cells requires two accessory factors in addition to the protease ClpXP: CpdR and RcdA. However, in vitro studies reveal that purified ClpXP is competent to degrade CtrA without CpdR or RcdA present, and addition of RcdA alone does not increase the rate of proteolysis. In the last year we have tested hypotheses to reconcile the discrepancy between in vitro and in vivo results. 1) We hypothesized that both CpdR AND RcdA might be necessary to increase the rate of CtrA degradation by ClpXP. We overexpressed and purified CpdR and added it, alone or along with RcdA, to our in vitro system. We found that CpdR alone or in combination with RcdA fails to stimulate CtrA proteolysis by ClpXP. Therefore, these proteins cannot act as a traditional adaptor that stimulates CtrA degradation. 2) We hypothesized that RcdA may be post-translationally modified in Caulobacter, affecting its activity. RcdA purified from Caulobacter
did not show any modifications by mass spectrometry. 3) We hypothesized that RcdA could inhibit a negative regulator of CtrA proteolysis which is present in cells but not in the in vitro assay. We performed a screen to detect negative regulators of CtrA proteolysis and isolated ~10 candidate mutants. An undergraduate is currently measuring the rate of CtrA proteolysis in these strains by an independent method to select mutants for further study. In collaboration with the Fletterick lab at UCSF, we determined the crystal structure of RcdA. RcdA is a dimer, and each monomer is three-helix bundle with three intrinsically disordered regions: the N-terminus, a loop connecting helices 2 and 3, and the C-terminus. We have made structure-guided mutations in rcdA to identify surface features necessary for CtrA degradation in vivo. Six substitutions at conserved amino acid positions did not impair CtrA proteolysis individually or together. In contrast, truncation of the molecule to remove the
C-terminal disordered peptide blocked CtrA proteolysis. This variant, RcdAΔC, failed to localize at the cell pole or midcell and also failed to promote CtrA localization to the cell pole at times when it is normally degraded. We are preparing this work for publication. Future studies will include additional structure-guided mutations and a yeast two-hybrid screen to identify RcdA-interacting proteins. The N-terminal receiver domain of CtrA is necessary for polar localization and proteolysis in cells, and a primary question is what factors recognize this domain? Purified ClpXP degrades CtrA, but it also degrades a truncated protein lacking the receiver domain. Neither RcdA nor CpdR binds directly to CtrA in vitro. We are using a yeast two-hybrid screen to identify unknown proteins that bind the CtrA receiver domain and target it to the cell pole for degradation. We hypothesized that the essential kinase DivL could regulate CtrA proteolysis via the CckA-ChpT-CpdR phosphorelay. We
performed pulse-chase analyses in divL and other mutants to measure CtrA degradation and determine the role of DivL in this process.
PARTICIPANTS: Kathleen Ryan, PI. Designed experiments, interpreted data, trained students and postdocs. Sarah Reisinger, graduate student in Ryan lab. Designed and performed experiments to measure CtrA proteolysis in various Caulobacter mutants. James Taylor, postdoctoral researcher in Ryan lab. Made and analyzed site-directed mutants of RcdA. Performed in vitro proteolysis experiments. Stephen Smith, rotation student in Ryan lab. Performed screen for Caulobacter mutants lacking a negative regulator of CtrA proteolysis. Jeremy Wilbur, Fletterick lab, UCSF. Purified and solved crystal structure of RcdA. Two graduate students and one postdoc in my lab have received training while working on this project. In particular, James Taylor has learned techniques in protein biochemistry and protein localization in bacteria. Sarah Reisinger learned to do pulse-chase analyses to measure CtrA protein half-life in wild-type and mutant Caulobacter strains. Sarah Reisinger presented her work at the
American Society for Microbiology general meeting in 2007 and published a paper including these results. Stephen Smith learned basic techniques for manipulating Caulobacter strains and screening for mutants affected in CtrA degradation.
TARGET AUDIENCES: For the community of scientific researchers: published a study including the effects of DivL on CtrA proteolysis. Sarah Reisinger presented an abstract at the ASM general meeting. The PI delivered a seminar on RcdA structure and function at MIT in November, 2007. For graduate students: the PI developed a new course module for first-year graduate students in microbiology about microbial genetics. Some examples of genetic screens were taken from our research on CtrA proteolysis. For undergraduate students: the PI continued to teach half of General Microbiology, an upper-level undergraduate course for >100 students. Some examples of genetic techniques and protein localization in bacteria were drawn from our studies of CtrA regulation.
Impacts
RcdA and CpdR together do not act as a traditional adaptor that stimulates CtrA degradation. It is still possible that they perform this function along with another, unidentified component. We have not identified any post-translational modifications that affect RcdA activity in Caulobacter cells. We have obtained a crystal structure of RcdA. It represents a new three-helix bundle fold with three intrinsically disordered domains. One of the disordered regions, the C-terminal peptide, is required for RcdA localization, CtrA localization, and CtrA proteolysis in Caulobacter. DivL is required to maintain normal levels of CtrA phosphorylation, but does not modulate the rate of CtrA degradation.
Publications
- Reisinger, S.J., Huntwork, S., Viollier, P.H., and Ryan, K.R. (2007). DivL performs critical cell cycle functions in Caulobacter crescentus independent of kinase activity. J. Bacteriol. 189: 8308-8320.
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Progress 01/01/06 to 12/31/06
Outputs
We are currently performing two genetic screens for mutants in which the CtrA protein cannot be proteolyzed. In one screen, we have identified several amino acid changes in the CtrA protein itself that block its degradation in vivo. We anticipate that these mutations will define the binding site for an adaptor protein that delivers CtrA to the ClpXP protease. In the other screen, we have constructed a library of PCR-mutagenized rcdA genes which we are testing for their ability to promote CtrA degradation in vivo. We determined that our previous protocol for purifying the ClpX ATPase did not yield fully functional protein. We are therefore testing new purification methods in order to assess degradation in vitro of the ctrA mutants we have obtained. We will also test any rcdA mutants we obtain for their ability to bind to ClpXP, stimulate the ATPase activity of ClpX, or promote degradation of the wild-type CtrA protein. Finally, in collaboration with the Fletterick lab
at UCSF, we are crystallizing the RcdA protein to determine its structure. Biochemical data suggest that RcdA does not interact directly with CtrA, so it may not function as an adaptor protein making a ternary complex with the protease and substrate. Since RcdA has no homologs of known function, we hope that the three-dimensional structure along with the biochemical experiments described above will shed light on its biological role. In a related collaboration with the Laub lab at Harvard University (currently at MIT), we found that the CckA-ChpT phosphorelay which activates CtrA also controls its proteolysis by phosphorylating a second response regulator, CpdR. When CckA and ChpT are active, both CtrA and CpdR are phosphorylated, and CtrA is active and stable. When the phosphorelay is inactive, both CtrA and CpdR are dephosphorylated, and CpdR promotes CtrA degradation. Another essential cell cycle regulator, DivK, modulates the activity of the CckA-ChpT phosphorelay, accounting for
the differential stability of CtrA in the stalked and swarmer progeny of Caulobacter cell division. In future studies, we will assess the biochemical role of CpdR, along with RcdA, in promoting CtrA proteolysis.
Impacts
Our work has identified a regulator of CtrA proteolysis (RcdA) that is conserved the closely related alpha proteobacteria Agrobacterium tumefaciens, Sinorhizobium meliloti and Brucella abortus. Since CtrA has been shown to be important for the normal life cycles of these bacteria, RcdA may also be an important regulator in these agriculturally significant bacteria. We have also shown that the ClpX subunit of the ClpXP protease is localized to specific regions within the Caulobacter cell. Most prokaryotes have Clp proteases, and they are essential in some bacteria. This is the first indication that these ubiquitous proteases are not simply dispersed throughout the cytoplasm. Clp protease localization may be an important regulator of cell behavior across many bacterial species.
Publications
- McGrath, P.T., Iniesta, A.A., Ryan, K.R., Shapiro, L., and McAdams, H.H. (2006). A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator. Cell 124:535-547.
- Biondi, E.G., Reisinger, S.J., Skerker, J.M., Arif, M., Perchuk, B.S., Ryan, K.R., and Laub, M.T. (2006). Regulation of the bacterial cell cycle by an integrated genetic circuit. Nature 444: 899-904.
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Progress 01/01/05 to 12/31/05
Outputs
We have initiated further studies of the RcdA protein required for cell cycle-regulated CtrA proteolysis in Caulobacter crescentus. During the cell cycle, levels of RcdA rise as CtrA is degraded. This finding led to the hypothesis that RcdA accumulation might trigger CtrA proteolysis. We placed rcdA under the control of a xylose-inducible promoter and demonstrated high levels of RcdA throughout the cell cycle do not disrupt the timing of CtrA proteolysis lead to more rapid degradation of CtrA than in wild-type cells. Therefore, other cellular mechanisms are required to specify the timing of CtrA proteolysis. RcdA, CtrA, and ClpX can be co-immunoprecipitated from Caulobacter lysates, suggesting that they form a complex within the cell. However, we have detected no interactions among the purified RcdA, CtrA and ClpX proteins in vitro. It may be that additional, unknown components are required to form the complex. To test this hypothesis, we are engineering a TAP (tandem
affinity purification) tag at the C-terminus of RcdA to facilitate the purification of RcdA-containing complexes from Caulobacter lysates. We will isolate complexes from native or chemically crosslinked lysates, as well as from cell lysates lacking the tagged version of RcdA, and compare the isolated proteins by SDS-PAGE. Protein bands that are specifically purified along with the tagged RcdA will be sent to the Berkeley mass spectrometry facility for peptide mapping and identification using the Caulobacter genome sequence. It is also possible that our purified Caulobacter ClpX protein is behaving differently than it does in the cellular context. The labs of Tania Baker and Bob Sauer have studied E. coli ClpX extensively in vitro. I am working with a postdoctoral fellow in the Baker lab, Peter Chien, to develop new purification strategies for Caulobacter ClpX that will preserve its native activity. Along the Shapiro and McAdams labs, we found last year that ClpX and RcdA are located
to the same intracellular positions during the division cycle. These positions include the sites of CtrA proteolysis, but also include localization to the midcell in early predivisional cells. These results suggest that RcdA may not act as a specific adaptor for CtrA proteolysis, but may have a more general role in ClpX substrate selection or activity. Since RcdA has no homologs of known function in sequenced genomes, we are purifying and crystallizing RcdA for structural studies in collaboration with Tom Alber at Berkeley. We anticipate that the molecular structure of RcdA will help us to generate hypotheses about RcdA function and will guide mutagenesis studies of RcdA itself. We are continuing to analyze the degradation and localization of point mutants within the CtrA receiver domain to determine which amino acids are critical for these processes. We anticipate that they will form a binding site for a proteolytic adaptor, which may or may not be RcdA.
Impacts
Our work has identified a regulator of CtrA proteolysis (RcdA) that is conserved the closely related alpha proteobacteria Agrobacterium tumefaciens, Sinorhizobium meliloti and Brucella abortus. Since CtrA has been shown to be important for the normal life cycles of these bacteria, RcdA may also be an important regulator in these agriculturally significant bacteria. We have also shown that the ClpX subunit of the ClpXP protease is localized to specific regions within the Caulobacter cell. Most prokaryotes have Clp proteases, and they are essential in some bacteria. This is the first indication that these ubiquitous proteases are not simply dispersed throughout the cytoplasm. Clp protease localization may be an important regulator of cell behavior across many bacterial species.
Publications
- McGrath, P.T., Iniesta, A.A., Ryan, K.R., Shapiro, L, and McAdams, H.H. 2006. A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator. Cell 124: 535-547.
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Progress 01/01/04 to 12/31/04
Outputs
In collaboration with the Shapiro lab at Stanford, we have identified a new protein, RcdA, which is required for the cell cycle-regulated proteolysis of CtrA in Caulobacter crescentus. Cells in which the rcdA gene has been replaced by a hygromycin resistance cassette (rcdA knockout) are viable, but they cannot proteolyze CtrA. Furthermore, a YFP-CtrA fusion protein does not localize transiently to the cell pole in cells lacking RcdA. A YFP-RcdA fusion protein complements the rcdA knockout and is dynamically localized during the cell cycle. At both times when CtrA is localized and degraded, during the swarmer-to-stalked cell transition and in the stalked compartment of the late predivisional cell, RcdA is present at the same subcellular location. These data strongly suggest that RcdA is the adaptor protein that mediates both CtrA localization and proteolysis during the Caulobacter cell cycle. To determine how RcdA promotes CtrA degradation, we have overexpressed and
purified RcdA, CtrA, ClpX, and ClpP. We will use these purified proteins to assess the following activities: 1) binding of RcdA to CtrA, 2) binding of RcdA to ClpX, 3) formation of a ternary complex of RcdA, CtrA, and ClpX in the presence of non-hydrolysable ATP analogs, 4) stimulation of ClpX ATPase activity by RcdA or RcdA plus CtrA, and 5) degradation of CtrA by ClpXP in the presence and absence of RcdA. To detect an interaction between RcdA and CtrA in living cells, we are constructing a strain containing both the GFP-CtrA and RcdA-RFP fusion proteins, which will be used to measure fluorescence resonance energy transfer (FRET) between the GFP and RFP fluors. While colocalization of two fluorescent proteins only demonstrates proximity up to the resolution limit of light microscopy, a FRET signal only occurs if two proteins are within 100 angstroms of each other, indicating a close functional interaction. Surprisingly, we have found that the ClpX subunit of the ClpXP protease is
dynamically localized during the cell cycle, in a manner qualitatively similar to RcdA. This observation suggests that ClpP may also be localized, and it also raises the question why ClpX and RcdA travel to the division plane in predivisional cells, before CtrA degradation at the stalked pole. We are currently examining the location of a ClpP-YFP fusion protein in Caulobacter to determine if it tracks with ClpX or is found at diverse sites within the cell, where it could also interact with ClpA. In previous work, we deduced a set of 11 amino acids that were likely to be required for CtrA proteolysis during the cell cycle. We have used site-directed mutagenesis to change all of these amino acids from the wild-type CtrA residues to those of CzcR, a homolog of CtrA from Rickettsia prowazekii that is not proteolyzed in Caulobacter. These mutations abolish CtrA turnover at the swarmer-to-stalked cell transition. We are currently making groups of 2-3 mutations to pinpoint the signal for
cell cycle-dependent proteolysis. We hypothesize that these residues may be involved in binding to RcdA, and we will use mutated CtrA proteins in the in vitro assays described above.
Impacts
Our work has identified a regulator of CtrA proteolysis (RcdA) that is conserved the closely related alpha proteobacteria Agrobacterium tumefaciens, Sinorhizobium meliloti and Brucella abortus. Since CtrA has been shown to be important for the normal life cycles of these bacteria, RcdA may also be an important regulator in these agriculturally significant bacteria. We have also shown that the ClpX subunit of the ClpXP protease is localized to specific regions within the Caulobacter cell. Most prokaryotes have Clp proteases, and they are essential in some bacteria. This is the first indication that these ubiquitous proteases are not simply dispersed throughout the cytoplasm. Clp protease localization may be an important regulator of cell behavior across many bacterial species.
Publications
- No publications reported this period
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