Identification of the Pba1 and Pba2 Binding Sites on 20S Core Particle Intermediates

Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Entitled For the degree of Is approved by the final examining committee: Chair To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________ ____________________________________ Approved by: Head of the Graduate Program Date Lindsay Jo Hammack Identification of the Pba1 and Pba2 Binding Sites on 20S Core Particle Intermediates Master of Science Andrew Kusmierczyk Stephen Randall Anna Malkova Andrew Kusmierczyk Simon Atkinson 07/09/2012 Graduate School Form 20 (Revised 9/10) PURDUE UNIVERSITY GRADUATE SCHOOL Research Integrity and Copyright Disclaimer Title of Thesis/Dissertation: For the degree of Choose your degree I certify that in the preparation of this thesis, I have observed the provisions of Purdue University Executive Memorandum No. C-22, September 6, 1991, Policy on Integrity in Research.* Further, I certify that this work is free of plagiarism and all materials appearing in this thesis/dissertation have been properly quoted and attributed. I certify that all copyrighted material incorporated into this thesis/dissertation is in compliance with the United States’ copyright law and that I have received written permission from the copyright owners for my use of their work, which is beyond the scope of the law. I agree to indemnify and save harmless Purdue University from any and all claims that may be asserted or that may arise from any copyright violation. ______________________________________ Printed Name and Signature of Candidate ______________________________________ Date (month/day/year) *Located at http://www.purdue.edu/policies/pages/teach_res_outreach/c_22.html Identification of the Pba1 and Pba2 Binding Sites on 20S Core Particle Intermediates Master of Science Lindsay Jo Hammack 07-09-2012 !"#$%!&!'(%!)$*)&*%+#*,-(.*($"*,-(/*-!$"!$0*1!%#1*)$*/21*')3#*,(3%!'4#** * !$%#35#"!(%#1* * * * (*%67898* * 1:;<9==7>*=?*=67*&@A:B=C* * ?D* * ,:E>:7*FG9H7E89=C* * ;C* * 49G>8@C*I?*+@<<@AJ* * * * !G*,@E=9@B*&:BD9BB<7G=*?D*=67* * 37K:9E7<7G=8*D?E*=67*"7LE77* * ?D* * 5@8=7E*?D*1A97GA7* * * * (:L:8=*/2./* * ,:E>:7*FG9H7E89=C* * !G>9@G@M?B98N*!G>9@G@* * * * 99* 99* ('O$)P4#"0#5#$%1* &9E8=*@G>*D?E7*<?8=N*!*Q?:B>*B9J7*=?*=6@GJ*"ER*(G>E7Q*O:8<97EASCJ*D?E*698* 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of  α and  one  type of  β  subunit    When  these  subunits  are  expressed   recombinantly  in  E.coli the  α and  β  subunits  will  self-­‐assemble  into  fully  active 20S   proteasomes  indistinguishable  from  their  natively  purified  counterparts    If  only  α   subunits  are  expressed,  these...  spermatogenesis  (Zhong  &   Belote,  2007)   The  function of the  other  testes-­‐specific  subunits  is  not  known     1.6 Activators     As  previously  mentioned,  once the 20S  CP  has  been  assembled the  N-­‐termini of   the  a  subunits  act  as  a  gate  closing  off the  catalytic  chamber    In  order  to  allow   substrates  to  enter the 20S  CP, the  gate  must  be  opened, and  in...       One of the  early  steps  in the  degradation of  a  protein  by the  ubiquitin-­‐ proteasome  pathway  involves the  modification of  this  protein  via the  covalent   attachment of  ubiquitin    A  single  ubiquitin  protein  is  roughly  8  kDa, and  it  is  usually   linked  to  proteins  via  an  isopeptide  bond  between the  carboxyl-­‐terminus of the   ubiquitin and  a...  to the  mammalian  orthologs,  yeast  proteasome  chaperone  proteins Pba1   and Pba2  have  been  characterized  as  a  heterodimer  that  is  found  exclusively on   proteasomal  precursors  (Li  et  al,  2007)    Often  times  these  precursors  contain the  Ump1   chaperone  protein    Little  is  known  about the  specific  function of the  Pba1p-­‐Pba2p    In   order  to  assess the. ..  to  these   chaperone  proteins  associated  with  α5 and  α7    However,  a  recent  study  by  Park  et  al,   (2011)  suggested  another  set of  α  subunits    Park and  colleagues  set  out  to  observe the   interface  between the  19S  RP and the  α-­‐ring of the 20S  CP    In  this  study  mutations   were  made  to the  intersubunit  pockets of the  α-­‐ring,  so  that the. .. of 20S  assembly  factors,  this  thesis  will  focus  specifically on the Pba1- ­ Pba2  protein   complex    Despite  two  different  studies  suggesting  that  Pba1p and  Pba2p  associate  with   specific  α  subunits,  α5 and  α7  (Hirano  et  al,  2005) and  α5 and  α6  (Park  et  al,  2011),  no   study  to  date  has  shown  definitively  where  Pba1p and  Pba2p  bind on the 20S  CP...  Pba2p  bind on the 20S  CP   intermediates    Recently,  a  group  revealed  how  Pba1p and  Pba2p  bind  to the 20S  CP     This  group  demonstrated  that  Pba1p and  Pba2p  contain  functional  C-­‐terminal  HbYX   motifs    These  HbYX  motifs  are  essential  for binding,  contribute  to  assembly  in  vivo, and   are  partially  redundant  with  one  another  (Kusmierczyk  et  al,  2011)...  plasmid-­‐borne  α  subunits  contains  a   C-­‐terminal  tandem  affinity  purification  (TAP)  tag, and the  plasmids  themselves  contain  a       23   URA3  gene,  enabling  selection on  SD-­‐Ura  media   The  X  residue of  Pba1p and  Pba2p   HbYX  motifs  were  both  mutagenized  to  cysteine  residues    In  brief, the  C-­‐terminal  end of   the  wild-­‐type PBA1  plasmid  (AKB495)  was... the  Rpts of the  19S and the  conserved  lysine  residue of the  α  subunits   would  be  disrupted    When  this  group  performed  mass  spectrophotometry on  purified   proteasome  precursor  complexes  isolated  from  these  lysine  mutants,  they  revealed  that   the  α5  mutant  had  significantly  less  Pba1p-­‐Pba2p  present, and  α6  mutant  had  very  little   Pba2p  present and. ..  et   al,  2012)    These  new  images of the  19S and the  26S  are  changing the  way the  field   views the  19S  regulatory particle   1.3.3 Archaeal and  Eubacterial 20S  Proteasomes     20S  proteasomes  have  been  identified  in  both  eubacteria and  archaea    Even   though the  first  proteasomes  were  identified  in  eukaryotes, the  prokaryotic  versions   have  been  used