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INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313 761-4700 800 521-0600 Order N u m b e r 9238146 Membrane assembly studies of subunit H, leader peptidase and M13 procoat proteins Escherichiain colt Cheng, Shiyuan, Ph.D. The Ohio State University, 1992 UMI 300 N. Zeeb Rd. Ann Aibor, MI4H106 MEMBRANE ASSEMBLY STUDIES OF SUBUNIT H, LEADER PEPTIDASE AND M13 PROCOAT PROTEINS IN ESCHERICHIA CPU DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By SHIYUAN CHENG. B. S. The Ohio State University 1992 Dissertation Committee: Approved by ^ * " * e “ i w Pappachan E. Kolattukudy Advisor James A. Cowan Ohio State Biochemistry Program*—•" To my wife, Bo Hu and my parents, for their continuous love, understanding and support. To my lovely daughter, Sarah (Ruirui), for bringing me wonderful joy. ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my mentor Dr. Ross E. Dalbey for his kind understanding, his great encouragement at the turning point of my graduate career, and for his generous moral and material support as well as for his thoughtful advice and guidance throughout the research. I would also give thanks to Dr. Samuel Kaplan for instructing me how to express the subunit H protein. My sincere appreciation also goes to my colleagues, Heng-Yi, Bill, Liming, Meesook, Lee, Guoqing, Xintong, Ruby, Songho and people in laboratories of Drs. Ming-Daw Tsai and James A. Cowan, for their friendship, helpful discussions and sharing instruments. Special thanks also to Drs. Andreas Kuhn and Gunnar von Heijne for their cooperation. Finally to my family, especially to my lovely wife, whose love, patience, understanding, encouragement, support and priceless gift of my beautiful daughter make me forever indebted. To Sarah (Ruirui) for being the world’s most wonderful and beautiful daughter. Thanks also to my parents and siblings for their continuous love, encouragement and support throughout my life . i i i VITA July 1, 1957 .......................................... Born in Wuhan, P. R. China January, 1982 .......................................... B.S. in Biochemistry, Wuhan University Wuhan, P. R. China September, 1985-Present ................... Graduate Research Associate or Teaching Associate in The Ohio State Biochemistry Program, The Ohio State University. PUBLICATIONS Shen, L. M., Lee, J. I., Cheng, S. Y., Jutte, H., Kuhn, A., and Dalbey, R. E. (1991) "Site-directed Mutagenesis to Define the Limits of Sequence Variation Tolerated for Processing of the M13 Procoat Protein by the Escherichia coli Leader Peptidase", Biochemistry, 30, 11775- 11781. i v Farooqui, A. A., Rammohan, K. W., Cheng, S. V., and Horrocks, L. A (1988) "Membrane Bound Diacylglycerol Lipase in Bovine Brain: Purification, Characterization, and Regulation" Frontiers of Chemistry: Vol. 1, Biotechnology. 75-89. Published by Chemical Abstract Services, Columbus, Ohio. Field of Study: Major Field: Biochemistry Emphasis in Molecular and Cell Biology, Professor Ross E. Dalbey, Advisor. v TABLE OF CONTENTS PAGE DEDICATION i i ACKNOWLEDGEMENTS i i i V ITA i v LIST of TABLES............................................................................................ ix LIST of FIGURES.......................................................................................... x LIST of ABBREVIATIONS........................................................................... x i v CHAPTER PAGE I INTRODUCTION ............................................................................. 1 1.1. The Role of Charged Amino Acid Residues in Membrane Protein Topology ......................................... 1 1.2. Subunit H Protein of the Photosynthetic Reaction Center from Rhodobacter sphaeroides ................... 11 1.3. Protein Export in £. coli .................................................. 15 1.4. £. coll Leader Peptidase ................................................ 2 2 11. MATERIALS AND METHODS.......................................................... 2 7 2.1. Materials............................................................................ 2 7 2.2. Bacterial Strains, Media and Plasmids .................... 2 9 2.3. Special Growth of the Cells Carrying Subunit H Protein Gene of the Photosynthetic Reaction Center from Rhodobacter sphaeroides .................. 3 0 2.4. Preparation of E. coli Cell Membrane Fractions. 31 v i 2.5. Alkaline Phosphatase Activity Assay ................... 3 3 2.6. Reverse Protease Mapping on Subunit H Protein Expressed in £. soli ........................................................ 3 4 2.7. Alkali Fractionation ........................................................ 34 2.8. SDS-PAGE Gel Electrophoresis and Fluorography 3 5 2.9. Agarose Gel Electrophoresis ....................................... 41 2.10. Polyacrylamide Gel Electrophoresis for DNA Sequencing Analysis ..................................................... 41 2.11. Preparation of M13 RF (Replicative Form) and Plasmid DNA ............................................................ 4 2 2.12. Oligonucleotide-Directed Mutagenesis ................. 4 5 2.13. DNA Sequencing ........................................................... 4 9 2.14. Subcloning .................................................................... 5 0 2.15. Transformation and Transfection ............................. 53 2.16. Screening for Positive Clones with Different Methods ......................................................................... 54 2.17. Preparation of Spheroplasts ........................................ 5 7 2.18. Protease Accessibility Assay ..................................... 59 2.19. Immunoprecipitation ....................................................... 6 0 2.20. Immunoblotting ............................................................... 61 III. THE USE OF GENE FUSION TO DETERMINE WHICH REGIONS OF A PROTEIN ARE IMPORTANT FOR THE MEMBRANE ORIENTATION OF SUBUNIT H PROTEIN OF THE PHOTOSYNTHETIC REACTION CENTER FROM RHODOBACTER SPHAEROIDES ....................................... 63 3.1. Introduction ................................................................ 6 3 3.2. Construction of Subunit H-PhoA Gene Fusions .. 6 7 3.3. Expression in £. coli of Subunit H Protein of the Photosynthetic Reaction Center from Rhodobacter sphaeroirifis ....................................................................... 7 6 3.4. The Insertion of Subunit H Protein into the Cytoplasmic Membrane of £. coli .............................. 7 7 3.5. Expression of the Subunit H-PhoA Fusion Proteins ............................................................................ 8 5 3.6. The Negatively Charged Amino Acid Clusters Are Not Determinants of Membrane Orientation of Subunit H Protein ..................................................... 8 6 3.7. Conclusion .................................................................... 91 v i i IV. SECB-DEPENDENT AND SECB-INDEPENDENT INSERTIONS OF£. COLI LEADER PEPTIDASE 9 6 4.1. Introduction ..................................................................... 9 6 4.2. SecB Is Required for the Complete Translocation of Altered Leader Peptidases .... 101 4.3. Conclusion ................................................................... 113 V. REQUIREMENT OF A MEMBRANE ANCHOR DEPENDS ON WHETHER OR NOT A PROTEIN IS SEC-DEPENDENT FOR MEMBRANE ASSEMBLY ............................................................... 1 1 8 5.1. Introduction ..................................................................... 118 5.2. Inverted Leader Peptidase Requires Its Second Hydrophobic Domain for Membrane Assembly .. 124 5.3. Other Substitutions with Arginines in H2 Region Did Not Affect the Kinetics of Inverted Leader Peptidase Membrane Insertion ................................... 12 7 5.4. The Membrane Anchor Region Is Not Essential for Sec-Dependent Procoat Membrane Insertion 129 5.5. Insertion of Inverted Leader Peptidase Mutants That Is Not Affected by a Positive Charge Is SecA-independent ...................................... 134