Houtz Lab

Welcome to the Houtz lab Home Page. Here, we tell you about the Research in the lab, the People who do it, and our Publications, as well as a brief desciption of Bob Houtz' Teaching Responsibilities and Educational Background.

Research Focus

 Our research is focused on providing a detailed enzymological, molecular, and functional analysis of the chloroplast-localized post-translational processing enzyme responsible for the site-specific methylation of Lys-14 in the LS of Rubisco. The formation of enzymatically catalyzed site-specific trimethyllysyl residues has significant effects on functional attributes of several other target protein substrates. Two well described examples are the site-specific methylation of Lys-77 in cytochrome c by a cytochrome c specific N-methyltransferase, which results in a 4-fold increase in import by isolated mitochondria, and methylation of Lys-115 in calmodulin by a calmodulin specific N-methyltransferase, which results in a 3-fold reduction in NAD kinase activating activity. The formation of trimethyllysyl residues in both cytochrome c and calmodulin has been reported to decrease suceptibility to proteolysis, by blocking a potential ubiquitination site in calmodulin, and by increased resistance to non-specific proteases in cytochrome c. However, there has been no clear unifying hypothesis with regards to the in vivo functional significance of covalent modification of the epsilon-amine group of specific lysyl residues in proteins by these highly specific protein N-methyltransferases. This may be in part do to the functional diversity of the target protein substrates, which encompass proteins with structural, regulatory, and enzymatic properties. Indeed it has even been proposed that protein methylase III enzymes may have co-evolved along with the respective protein substrates.

With no clear analogy between the functional affects of methylation in proteins such as cytochrome c and calmodulin, the functional role of site-specific methylation of Lys-14 in the LS of Rubisco is open to speculation. The mechanism by which protein methylase III enzymes exert such a high degree of specificity towards not only one specific protein, but only one or sometimes two lysyl residues within that target protein, is unknown. Also, untill the recent results from our laboratory there has been no available DNA or protein sequence for any protein methylase III enzyme. We previously proposed that an analysis of the functional significance of trimethyllysine in the LS of Rubisco may result in a significant contribution to the understanding of processes critical to functional aspects of Rubisco which are as yet unknown. Furthermore, we proposed that an approach which included purification of Rubisco LSMT would result in additional and equally significant contributions towards the molecular and biochemical characterization of a protein methyltransferase III enzyme, and potentially enable novel examinations of the in vivo functional significance of trimethyllysine in the LS of Rubisco through construction of transgenic plants and molecular perturbation of Rubisco LSMT activity. To date we have sucessfully purified Rubisco LSMT to homogeneity using a novel purification technique, obtained complete protein and nucleotide sequence for Rubisco LSMT, partially defined the interaction between Rubisco LSMT and des(methyl) Rubisco, and finally performed a thorough examination of the kinetic and enzymological consequences of methylation of Lys-14 in the LS of holoenzyme Rubisco.

Since the site of methylation in the LS of Rubisco resides in an area of the protein that is: 1) required for maximum levels of catalytic activity; 2) undergoes catalytic dependent and effector-mediated changes in conformation and solvent acessibility; and 3) lies next to a residue (Phe-13) whose side-chain has indirect contact with an active-site lysyl residue, we targetted our initial studies towards determining if methylation of Lys-14 affected Rubisco activity.

Our recent comparative enzymological studies of in vitro methylated and non-methylated forms of spinach Rubisco demonstrate that the methylation of Lys-14 in the LS of Rubisco has only a minor affect on a number of kinetic parameters. Given the relative irreversible nature of this covalent modification, these observations suggests that functional aspects of trimethylation of lysyl residue 14 in the LS of Rubisco are probably not related to the in vivo regulation of Rubisco activity.

However, methylation of Lys-14 by Rubisco LSMT does result in complete protection against proteolytic attack by trypsin and Lys-C endoprotease, a fact dramatically evident in studies comparing the loss in catalytic activity between methylated and non-methylated spinach Rubisco during limited proteolysis. This protection against in vitro proteolysis with purified proteases may not however, be related to Rubisco holoenzyme stability in vivo, since we have been unable to measure loss in catalytic activity (a sensitive measure of proteolytic cleavage at Lys-14 and/or adjacent residues in the N-terminus of the LS), or evidence of N-terminal proteolysis, when non-methylated spinach Rubisco is incubated in the presence of pea chloroplast lysates. However, we believe that these observations may represent a significant clue to the in vivo functional significance of trimethyllysine-14 in the LS of Rubisco. For example, recent studies with a synthetic polypeptide version of the LS of Rubisco from acetyl-Pro-3 to Tyr-25 demonstrated that the peptide bond between Lys-14 and Ala-15 was particularly sensitive to proteolytic cleavage during incubation with pea chloropolast lysates.

These recent accomplishments place us in a unique position to address the functional significance of Lys-14 methylation in the LS of Rubisco, by construction of transgenic plants expressing sense and antisense constructs of Rubisco LSMT. Furthermore, given the techniques we have developed for studying the binding of Rubisco LSMT to PVDF-immobilized des(methyl) Rubisco, our potential future access to recombinant Rubisco LSMT, the ability to utilize in vitro translated Rubisco LSMT to study uptake, processing (including N-terminal processing of Rubisco LSMT), and in situ post-translational processing (as in the case of uptake and processing of Rubisco LSMT by isolated chloroplasts from a des(methyl) Rubisco species like spinach), we should be able to unambiguously define the functional significance and biochemical role that trimethyllysine plays in the enzymology of the LS of Rubisco. Of equal importance will be information obtained relative to the mechanism by which Rubisco LSMT recoginizes the LS of Rubisco, and perhaps the targetting of this mechanism for manipulation through the design and synthesis of specific peptide inhibitors. These peptide inhibitors may have commercial ramifications as selective peptide herbicides directed against species with trimethyllysine in the LS of Rubisco and Rubisco LSMT activity.

People in the Lab

Teaching Responsibilities
ABT 201
Scientific Method and Logic in Agricultural Biotechnology, 1 credit hour, for Agricultural Biotechnology majors, designed to acquaint students with common experimental methods used in biotechnology.
HOR 375
Growth and Development of Horticultural Crops, 3 credit hours, team taught course, 8-11 lectures on water relations, post-harvest physiology, photosynthesis, and temperature stress tolerance.
HOR/AGR 622
Special Topics - Physiology of Plants, 3 credit hours, team taught course, 8-11 lectures, an in-depth examination of the biochemical mechanisms and enzymology associated with the assimilation of carbon by plants.
HOR 601
Special topics in Horticultural research, 1 credit hour, team taught course designed to give Horticulure students an appreciation of the diversity of basic and applied reserach programs in Horticulture.

Publications
  1. Houtz, R.L. and Royer, M. 1995 Characterization of the effects of enzymatically catalyzed site-specific methylation of Lys-14 in the large subunit of Rubisco. Biochemistry (in preparation).
  2. Wang, P., Royer, M., and Houtz, R.L. 1995 Affinity purification of Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit eN-methyltransferase Protein Expression and Purification 6:528-536.
  3. Klein, R.R. and Houtz, R.L. 1995 Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase Plant Mol. Biol. 27:249-261.
  4. Houtz, R.L., L. Poneleit, S.B. Jones, M. Royer, J.T. Stults. 1992. Post-translational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from several plant species. Plant Physiol. 98:1170-1174.
  5. Houtz, R.L., M. Royer, M.E. Salvucci. 1991. Partial purification and characterization of ribulosebisphosphate carboxylase/oxygenase large subunit eN-methyltransferase. Plant Physiol. 97:913-920.
  6. Houtz, R.L., R.M. Mulligan. 1991. Catalytic protection of tryptic sensitive sites in the large subunit of ribulosebisphosphate carboxylase/ oxygenase. Plant Physiol. 96:335-339.
  7. Knavel, D.E., R.L. Houtz. 1990. Characteristics of 'Main Dwarf' short-internode muskmelon genotype as compared with its normal-internode "parent" and F1 hybrid ('Main Dwarf' x 'Mainstream'). HortScience 25:1277-1279.
  8. Houtz, R.L., J. Stults, R.M. Mulligan, N.E. Tolbert. 1989. Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase. Proc. Natl. Acad. Sci. USA 86:1855-1859.
  9. Biernbaum, J.A., R.L. Houtz, S.K. Ries. 1988. Field studies with crops treated with colloidally dispersed triacontanol. J. Amer. Soc. Hort. Sci. 113:679-684.
  10. Mulligan, R.M., R.L. Houtz, N.E. Tolbert. 1988. Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: The amino-terminal residue of the large subunit is acetylated proline. Proc. Natl. Acad. Sci. USA 85:1513-1517.
  11. Houtz, R.L., R.O. Nable, G.M. Cheniae. 1988. Evidence for effects on the in vivo activity of ribulose-bisphosphate carboxylase/oxygenase during development of Mn toxicity in tobacco. Plant Physiol. 86:1143-1149.
  12. Nable, R.O., R.L. Houtz, G.M. Cheniae. 1988. Early inhibition of photosynthesis during development of Mn toxicity in tobacco. Plant Physiol. 86:1136-1142.
  13. Archbold, D.D., R.L. Houtz. 1988. Photosynthetic characteristics of strawberry plants treated with paclobutrazol or flurprimidol. HortScience 23:200-202.
  14. Sterling, T.M., R.L. Houtz, A.R. Putnam. 1987. Phytotoxic exudates from velvet leaf (Abutilon theophrasti) glandular trichomes. Amer. J. Bot. 74:543-550.
  15. Cockfield, S.D., D.A. Potter, R.L. Houtz. 1987. Chlorosis and reduced photosynthetic CO2 assimilation of Euonymus fortunei infested with Euonymus scale (Homoptera: Diaspididae). Environ. Entomol. 16:1314-1318.
  16. Houtz, R.L., S.K. Ries, N.E. Tolbert. 1985. Effect of triacontanol on Chlamydomonas. I. Stimulation of growth and photosynthetic CO2 assimilation. Plant Physiol. 79:357-364.
  17. Houtz, R.L., S.K. Ries, N.E. Tolbert. 1985. Effect of triacontanol on Chlamydomonas. II. Specific activity of ribulose-bisphosphate carboxylase/oxygenase, ribulose-bisphosphate concentration, and characteristics of photorespiration. Plant Physiol. 79:365-370.
  18. Houtz, R.L. and S.K. Ries. 1983. Triacontanol levels in ascending sugar maple sap. HortScience 18:101-102.
  19. Ries, S.K. and R.L. Houtz. 1983. Triacontanol as a plant growth regulator. HortScience 18:654-662.

Educational Background
PhD
1984, Michigan State, "Stimulation of Growth and Photosynthetic Carbon Metabolism in Chlamydomonas reinhardtii with Triacontanol"
MS
1980, Michigan State, "Development and Characterization of an In Vitro System Responsive to 1-Triacontanol"
BS
1977, Florida. 
Robert L. Houtz
	Department of Horticulture and Landscape Architecture
	N-318d Agricultural Science Center North
	University of Kentucky, Lexington, KY  40546
	859/257-1982 - Office	859/257-3376 - Lab
	859/257-2859 - FAX	rhoutz@uky.edu
Last modified Mar 29, 1996. Background: Turbina corymbosa at Cornell.