Carlson a DISSERTATION Presented To

Carlson a DISSERTATION Presented To

Mechanism of CFTR Dislocation During Endoplasmic Reticulum-associated Degradation by Eric J.;Carlson A DISSERTATION Presented to the Department of Cell & Developmental Biology and the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2006 School of Medicine Oregon Health & Science University CERTIFICATE OF APPROVAL This is to certify that the Ph.D. dissertation of Eric J. Carlson has been approved Me Member Member Member Member -- Table of Contents Acknowledgements ............................................................................... .iv Abstract. ............................................................................................ v Chapter 1. Introduction .......................................................................... 1 Cystic fibrosis ............................................................................... 2 Endoplasmic reticulum-associated degradation ........................................ 6 Strategy ...................................................................................... 15 Chapter 2. Identifying components of CFTR ERAD by biochemical manipulation of cytosol and rough microsomes ................................................................ 22 Abstract ...................................................................................... 23 Introduction ................................................................................. 24 Results ....................................................................................... 25 Discussion ................................................................................... 29 Methods ...................................................................................... 31 Chapter 3. Uncoupling Proteasome Peptidase and ATPase Activities Results in Cytosolic Release of an ER Polytopic Protein ................................................52 Abstract ......................................................................................53 Introduction ................................................................................. 54 Results ....................................................................................... 57 Discussion ................................................................................... 62 Methods ...................................................................................... 67 Chapter 4. p97 Functions as a Non-Essential Auxiliary Factor for TM Domain Extraction during CFTR ER-Associated Degradation .................................... 85 Abstract. ..................................................................................... 86 Introduction ................................................................................. 87 Results ....................................................................................... 90 Discussion ...................................................................................98 Methods .................................................................................... 103 Supplemental information ............................................................... 124 Summary ........................................................................................... 126 Future Studies .................................................................................... 128 References .......................................................................................... 13 2 Appendices ......................................................................................... 146 A. Preparation of RRL ................................................................... 147 B. Preparation of ER microsomal membranes ........................................ 152 C. In vitro transcription/translation ..................................................... 155 D. In vitro degradation and membrane release assays ............................... 159 E. Affinity adsorption ofRRL ERAD components .................................. 166 F. Inactivation and reconstitution ...................................................... 174 ii Table of Figures Figure 1-1. ABC transporter structure ........................................................ 17 Figure 1-2. CFTRfolding ...................................................................... 19 Figure 1-3. Dislocation and degradation of CFTR ..........................................21 Figure 2-1. Reconstitution of SRP-dependent translocation .............................. 38 Figure 2-2. Reconstitution of CFTR Biogenesis .............................................40 Figure 2-3. CFTR degradation into TCA soluble fragments ............................. .42 Figure 2-4. CFTR degradation in affinity depleted RRL ...................................44 Figure 2-5. CFTR degradation is decreased ..................................................46 Figure 2-6. LC-MS-MS of Ub-UbcH5a eluates ............................................ .48 Figure 2-7. RRL depletion by Ub-UbcH5a .................................................. .49 Figure 2-8. RRL depletion of p97 and p97 complexes ...................................... 50 Table 3-1 ...........................................................................................74 Figure 3-1. Cytosolic release ofTCA-insoluble CFTR fragments .........................75 Figure 3-2. Release of TCA-insoluble fragments is proportional. .........................76 Figure 3-3. Released CFTR fragments are heterogeneous ................................. 77 Figure 3-4. CFTR degradation involves simultaneous loss of multiple epitopes ........78 Figure 3-5. Cytosolic fragments are derived .................................................. 80 Figure 3-6. Cytosolic CFTR fragments remain stably associated .......................... 81 Figure 3-7. p97 is inhibited by hemin .......................................................... 83 Figure 4-1. CFTR is degraded into TCA soluble peptides ................................. 108 Figure 4-2. RRL depletion of p97 and p97 complexes ...................................... 11 0 Figure 4-3. p97 is effectively depleted from RRL ........................................... 112 Figure 4-4. Degradation of CFTR cytosolic domains ....................................... 114 Figure 4-5. P97 augmented degradation of a CFTR TM domain .......................... ll6 Figure 4-6. p97 effects are influenced ......................................................... 118 Figure 4-7. p97 directly facilitates membrane extraction .................................... 120 Figure 4-8. P97 effect is inversely related ..................................................... 122 Suppl.Figure 4-1 ................................................................................... 123 Figure S-1 .......................................................................................... 130 Figure E-1. Affinity depletion of RRL ......................................................... 172 Figure F-1. Trypsinization of microsomal membranes ...................................... 178 Figure F-2. Reconstitution of SRP dependent translocation ................................ 179 iii ACKNOWLEDGEMENTS To my wife and children for their patience all these years, and to everyone who helped along the way. Many thanks. iv Abstract Cystic fibrosis is a lethal recessive disorder caused by inherited mutations in the cystic fibrosis transmembrane conductance regulator (CFfR). Sixty to eighty percent of wild type CFfR is degraded, and the most common mutation, deletion of F508, leads to essentially complete degradation by the proteasome. Therefore, a detailed understanding of the degradation pathway would provide useful information for developing therapeutics. To identify components in CFfR degradation, two compartments were examined: membrane-localized components and cytosolic components. Inactivation of membrane proteins had no effect on CFfR degradation suggesting that either membrane proteins were not sensitive to treatment or that cytosolic proteins with redundant functions exist. In contrast, affinity depletion of rabbit reticulocyte lysate (RRL) using nonhydrolyzable ubiquitin fusion proteins containing E2 ubiquitin conjugating enzymes (Ub-UbcHSa (uu5) and Ub-Ubc6 (uu6)) decreased CFfR degradation significantly. Adsorbed proteins eluted from uu5 beads were identified by liquid chromatography­ tandem mass spectrometry (LC-MS-MS), and the most abundant protein identified was the AAA-ATPase, p97. Adaptor proteins mediate the diverse functions of p97, which range from membrane fusion to degradation. The heterodimer ufdllnpl4 binds ubiquitinated substrates and directs p97 to the degradation pathway where p97 has been proposed to segregate components of protein complexes, provide the driving force for dislocation and serve as an intermediary in presenting substrates to the proteasome. Given its proposed role in ERAD, p97 was chosen for further characterization in CFfR degradation. Depletion of RRL p97 resulted in a 50% reduction in both the rate and extent of CFfR degradation. However, p97 depletion had no effect on the degradation of v cytosolic domains but specifically stimulated degradation of transmembrane domains (TMDs). Moreover, the stimulatory effect was dependent on TMD hydrophobicity and stability. This effect was also correlated with the rate of degradation, suggesting that p97 functions as an accessory factor to the proteasome and enhances the rate and efficiency of thermodynamically stable

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