Encrypted Document Analysis, Thesis of Biological Sciences

Download Encrypted Document Analysis and more Thesis Biological Sciences in PDF only on Docsity! Role of the AAA protease Yme‘1 in folding of proteins in the mitochondrial intermembrane space Bernadette Schreiner Munchen 2012  Role of the AAA protease Ymel in folding of proteins in the mitochondrial intermembrane space Dissertation zur Erlangung des Doktorgrades der Fakultat fiir Biologie der Ludwig-Maximilians-Universitaét Miinchen vorgelegt von Bernadette Schreiner aus Limburgerhof Miinchen 2012 INTRODUCTION TABLE OF CONTENTS 1. INTRODUCTION 1.1 Cellular protein quality control systems 1.1.1 Proteins - the worker molecules of the cell 1.1.2 Protein quality control and homeostasis (proteostasis 1.1.3 Failure of protein quality control and homeostasis .... 1.2 Molecular chaperones 1.2.1 The Hsp70 system . 1.2.2 The chaperonins . 1.2.3 The Hsp90 system . 1.2.4 The Hsp100 system 1.3 Proteolytic systems 1.3.1 The AAA protein family . 1.3.1 The AAA protease family 1.3.2 LON proteases 1.3.3 ClpP proteases 1.3.4 FtsH proteases 1.4 Mitochondrial biogenesi: 1.4.1 Mitochondrial subcompartmentalization. 1.4.2 Mitochondrial protein import.... 1.5 Mitochondrial protein quality control. 1.5.1 Protein quality control in the mitochondrial outer membrane . 1.5.2 Protein quality control in the mitochondrial matrix 1.5.3 Protein quality control in the mitochondrial inner membrane . 1.5.4 Protein quality control in the mitochondrial intermembrane space 1.6 Mitochondrial m-and i-AAA protease. 1.7 Aim of the present study 2. MATERIALS AND METHODG........ccsssssscscsssnsessensenssnsescsncsneasessesseneessenceneeneeee I 2.1 Molecular biology methods. 2.1.1 Strategies for isolation of DNA. 2.1.2 Enzymatic editing of DN. 2.1.3 DNA purification and analysi 2.14 E. coli strains 2.1.5 Plasmids and cloning strategies. 2.2 Yeast genetic methods 2.3 Protein biochemistry methods 2.3.1 Analytical methods... 2.3.2 Preparation of proteins . 2.4 Cell biology methods 2.4.1 NaOH cell disruption 2.4.2 “Rédel’s” cell disruption. 2.4.3 “Fast Mitoprep” 2.4.4 “Big Mitoprep” 2.4.5 Generation of mitoplasts . 2.4.6 Digitonin fractionation of mitochondri: 2.4.7 Protease treatment . 2.4.8 Aggregation assay . 2.4.9 Ni-NTA agarose pulldown      !"#$"%#&'()*+%,'-%#./$#/)$#--0$%*1+2%.+2#.%.340$+2#$)*+%,'-%#.0.#-%*+2%..+0-5336#*#$)+%'*'(./#7%(%7)*+%.#$)%*$),,%+.889#+#7+%'*'(/$'+#%*.'**%+$'7#:0:'.#;#;,$)*#.,5%;;0*'<.+)%*%*1 =>?@ A B).../#7+$';#+$5'(#:0+%'*($)7+%'*.'(C%<CDE)1)$'.#/0:-'&*AF-#*+%(%7)+%'*'()11$#1)+%*1/$'+#%*.,5GFHEI)*-;).../#7+$';#+$5A JKLMN? 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MATERIALS AND METHODS 2.1 Molecular biology methods 2.1.1 Strategies for isolation of DNA 2.1.1.1 Isolation of genomic DNA from S. cerevisiae For isolation of genomic DNA, an overnight culture of the respective yeast strain was incubated at 30 °C while shaking (130 rpm) (Rose et al, 1990). Cells were pelleted by centrifugation (2500 x g, 5 min, RT), washed with 20 ml HO and disrupted by resuspension in 1ml breaking buffer (100 pg/ml zymolyase 20T, 1M sorbitol, 100 mM EDTA). After incubation for 1 h at 37 °C, the cells were washed with 1ml 1 M sorbitol and 100 mM EDTA, pelleted by centrifugation, resuspended and then incubated in 1 ml lysis buffer (50 mM Tris/HCl, 20 mM EDTA, 1 % (w/v) SDS, pH 7.5) for 30 min at 65 °C. The cell solution was supplemented with 400 11 5 M potassium acetate and incubated for 1 h on ice. DNA was separated from the protein fraction by centrifugation (20000 x g, 15 min, 4 °C). The supernantant was transferred to a new tube, DNA precipitated with isopropanol and pelleted by centrifugation (37000 x g, 10 min, 2 °C). After washing with 70 % ethanol, the DNA pellet was dried for 10 min at RT and resuspended in 100 pl sterile H,O. After determination of concentration with a Nanodrop 2000c spectrophotometer (Promega), the DNA was stored at -20 °C. 2.1.1.2 Isolation of plasmid DNA from E£. coli Plasmid DNA was isolated in small scale or large scale from E. coli based on the principle of alkaline lysis (Birnboim and Doly, 1979) using the “PureYield” Plasmid Midiprep System (Promega). E. coli clones containing the appropriate plasmid were inoculated in 50 ml LB4™? medium and grown overnight at 37 °C with shaking (130 rpm, Infor cell shakers). Bacteria were harvested by centrifugation for 5 min at 5000 rpm in JA-20 rotor (Beckman centrifuge) and resuspended in 6 ml “Cell Resuspension Solution“ (10 mM EDTA (pH 8), 50 mM Tris/HCl (pH 7.5), 100 pg/ml RNase A). 6 ml “Cell Lysis Solution“ (0.2 M NaOH, 1 % (w/v) SDS) and in a second step 10 ml “Neutralization Solution“ (4.09 M guanidine hydrochloride (pH 4.2), 759 mM potassium acetate, 2.12 M      !"#$%$&'()*+,-./012-3"4"5"467789:;<=>? @AB CDE4 FGHIJK>LMN>LJOPJ=QR>IOSTUMVGOWMTXOSTUMQGO;MPXOS=M;YZONUKGO[\G E]̂"_4̀ àb̂34cdefg QhiI;P<PjkUlImnKQiPQiopM= ?  D_4̀qrsb4]̂ltj9uuOUlIvGwOKJ;VGOJTIVGOpxKVyzONU>{GOKJ=VGO=M;{Oh|}jK<VQXO=M;~=M;Y€[GOkTG‚nNJNKoƒ ?„_… 9:;<=>DE4_8 77D†4‡r4 ˆBA"„ˆBB_9:;<=>̂b34cdefgsb4]ˆ8 "‰̂b34cdefgBA‰ŠC‹ŒBA ˆA=M;ŽB"‰Bb 6B‰ˆ7B B"sb4]ˆB=M;B"Œ …‰@ 7   8B"3"4"5"3†79:;<=>9:;<=>88b̂  BˆˆfŠ† 4f‡Bc…g"]8 B AB A"„BB_9:;<=>B8 4‘48qŠ’A_B]“   ]q‡†"b̂ cb]̂g”b̂ ŒB”b̂ • 4‡”ˆ]AB_a”ˆ]A_a”‹†–B‘4‡‡—”B”A MATERIALS AND METHODS For preparation of the according agar plates, 2 % (w/v) agar was added before autoclaving and plates were poured when the medium was cooled down to ca. 60 °C. 2.1.4.4 Preparation of electro-competent E£. coli cells 1 ml of 50 ml overnight E. coli culture was inoculated into 500 ml LB medium and incubated at 37°C in a shaker until an ODgoo of ca. 0.5 was reached. The culture was incubated on ice for 30 min followed by sedimentation through centrifugation for 5 min at 5000 rpm (JA-10 rotor, Beckman Coulter) at 4 °C. The cell pellet was washed with 500, 250 and 50 ml of ice-cold, 10 % (v/v) glycerol solution. The pellet was resuspended in 500 pl 10 % glycerol, and 40 pl aliquots were stored at -80 °C. 2.1.4.5 Transformation of E. coli with plasmid DNA by electroporation One ul of ligation reaction or of 1:100 diluted plasmid DNA was added to a 40 yl solution of electro-competent cells (see 2.1.4.4). The mixture was transferred to an ice- cold 2 mm electroporation cuvette, put into “GenePulser” (Bio-Rad) electroporator and transformed by a singular short voltage pulse at 2.5 kV/ 400 Q/ 25 uF. Afterwards, the cells were immediately diluted in 1 ml pre-warmed LM medium, transferred to a 1.5 ml reaction tube and incubated for 30 min at 30 °C shaking at 130 rpm. Cells were pelleted, resuspended in 100 pl of residual supernatant and plated on LB plates supplemented with the required antibiotics. The plates were incubated over night at 37 °C and ca. 15 single colonies analyzed by diagnostic PCR. 2.1.5 Plasmids and cloning strategies 2.1.5.1 Overview of plasmids used for expression in E. coli Plasmid Reference pET28a+-Ymel_AAA-Hisg This thesis pMAL-cRI-Imp1 This thesis      !"#"$!%&'()*+++,-#./#"0!123456789:;<9=>9::<?@=A>B>C=:=>>9DEFGHIJ<A>KFFFK=:A>KFFFKLC>:9:8CM7NOG9PCL9QELR<C>=8C>:@9::ST@<UV!W:XY!W9A>KFFFK= Z[\]]]]]̂ _̀ ̀ `̀ ̀ _̀___̂ ^̂_̂]̂_]̂ ]̂ ̀\a[A>KFFFKL Z[\]]]]_]̀ ^̀]___]_]_]̂ ]̀̂]]]\a[b !"#"$!%c'1)#"0!1d+e,Vfc789:;<9=W>CB>C=:=>>9DEFGHIJ<W>CKgK=:W>CKgKLC>:9:8CgFh@9JWG9PCL9QEMiR:B>=8C>:@9::giI@<j.0W:-#"kWWW9W>CKgK= Z[\]]]]_̀]̂ ̀̂]_̀ ^̀ ̂ ]̀ ̂ `̀ ̀ `̂ ̀ ^̂_]\a[W>CKgKL Z[\]]]]]]̂ ^̀]___]̂ ̀ __̀]_]__̂ ]̀]_̀]̂]_̂]_̂ \̀a[lmLLB=C>:<:==>noV(p(q#.#(A J=9CAMr@HGQ@?sRDStJu7QvWgr@DStJu7R ICL@w9(0 ox?CAMr@HGQ@?syz{RDStJu7Qv>P@DStJu7R 788CAMr@HGQ@?sRDStJ><Qs|}|}{RQvWgr@DStJ><R 788CAMr@HGQ@?syz{RDStJ><Qs|}|}{RQv>P@DStJ><R 788CAMr@HGQ@?sRDStJu7S{QvWgr@DStJu7SR ICL@9~9x?CAMr@HGQ@?syz{RDStJu7S{788 MATERIALS AND METHODS 2.2.2 Homologous recombination in S. cerevisiae a) Deletion of YMEI gene YMEI was deleted by homologous recombination with the corresponding PCR product, amplified from pFA6KANMX4 (Wach et al., 1997) using Ymeldelta_for and Ymeldelta_rev primers in the haploid yeast strain YPH499. To select for positive clones, transformed yeast cells were grown on medium containing kanamycin. Homologous recombination was confirmed by PCR, and the absence of Ymel by “Fast Mitoprep”. Ymeldelta_for 5’-TAA TTA TAA TAC ATT GTG GAT AGA ACG AAA ACA GAG ACG TGA TAG CGT ACG CTG CAG GTC GAC-3' Ymeldelta_rev 5'-GTC TTG AGG TAG GTT CCT TCA TAC GTT TAA CTT CTT AGA ATA AAA ATC GAT GAA TTC GAG CTC-3’ c) Chromosmal His7-tagging of C-terminus of Ymel Ymel_His_for primer, consisting of the last 45 3’ bases of YME/ and 18 bases of pYM5S plasmid, and Ymel_His_rev primer, consisting of the 45 first bases of YME1 3’- UTR followed by 18 further bases of pYM10 plasmid, were used to amplify a PCR fragment from pYM10 containing 5’ to 3’ the following sequences: the last 45 3” bases of YME1, His7-coding sequence, HIS cassette and 45 bases of the beginning of the 3’-UTR. The PCR fragment was transformed into the YPH499 wild-type strain, and homologous recombination confirmed by PCR. Transformants were selected on medium lacking histidine. Ymel_His_for 5'-GAT ATA GGC GAT GAT AAA CCC AAA ATT CCT ACA ATG TTA AAT GCA CAC CAT CAC CAT CAC CAT CAC-3' Ymel_His_rev 5'-GGT GTT ATG AAG CAA AAG CGA AAC CGA CCA GAA AAG AAC AAA GCA TTC ATC GAT GAA TTC GAG cTc G-3' b) Deletions of MGR1 and MGR3 genes MGR1 and MGR3 genes were deleted by homologous recombination in the haploid yeast strain YPH499 with the corresponding PCR products amplified by Mgrldelta_for and Mgrldelta_rev or Mgr3delta_ for and Mgr3delta_rev primers. PCR products      !!!!"!#$!%# $$!%&'()&'(#*+#%!,#!-!%# $!*.#$!/-!%/0123-4$56%4$56,!4$76%4$76,!*4$5#6% 89:;<=;;=;;=;;<==;;;=;=;;====;;<<==<;;>=<<=<<<<>;>=<;>;=>;<>>=;><;:?94$5#6, 89:<<>><===<<=<=<;>;<;>>=<;<<;=<<>;<<=;;>;<<<><;<=;><=><<==;><>;=;:?94$7#6% 89:;<>><<><=;=;<>===<<;<>>;=<<<<>=;;;=;;====;>>=;>=<;>;=>;<>>=;><;:?94$7#6, 89:==><<<=<===<==<=====>=;==;;===<===;;===<==>=><=;><=><<==;><>;=;4$56% 89:==;<<<;<;<<=<>==>==;>:?94$56, 89:=;=;;<<<>>>;<<<><<<;;:?94$76% 89:<<;=>==;<==>;===>==;;:?94$76, 89:======;<<<==>>>===<>>:?9@ABCDEFEGFHIFJ@KLHMMNOMEPLCQBKLQDFNORGEP&SF)45606%T!!$%#!UV7W/!!%&X&)5Y/!!%Z4V#!T4567W[\]36,T!!$%UV%!/!!%41457W[\]3%#-/05Y%/!!%Z4V#!T-!#%0123%$%Z4V$VW7W%#-$!"!̂#!UV7W/!!%4145T0[$!"T._+!!UV/!!%/$$%7W[\]3*]123%$-!!%Z1.Ù ̀-#[0!T#$!/%/0123*]!%!-!## $!*45606% VW[ab]2222abb]baab2a]aaab]bb]bab]b]]ba2baaba2a]]2b]a2b2]b2abb]2ba2[7W4567W[\]36, VW[2a]a]]b]b]aaba]a]bab]aaaaaaabbaaa2baaaa]a]b]2a]2ba]baa]]2bab2]2[7W     !"#$%& '%&()*++,#&-./01.12345)6'7809:;.5)6'784 8!!&()*++,#&-./01.123<=1+45)6'7809".5)6'784 8!!&()*++,#&-./01.12345)6'>03?*?*+409:;.5)6'>03?*?*+44 8!!&()*++,#&-./01.123<=1+45)6'>03?*?*+409".5)6'>3?*?*+4 8!!&()*++,#&-./01.12345)6'78)09:;.5)6'78)4 8!!&()*++,#&-./01.123<=1+45)6'78)09".5)6'78)4 8!!&()*++=&;-1@ABC;D*,#&-./01.12345)6'7809:;.5)6'784 8!!&()*++=&;-1@ABC;D*,#&-./01.123<=1+45)6'7809".5)6'784 8!!&()*++=&;-1@ABC;D*,)E.&1,#&-./01.12345)6'7809:;.5)6'784 8!!&()*++=&;-1@ABC;D*,)E.&1,#&-./01.123<=1+45)6'7809".5)6'784 8!!&()*++&1.)E,#&-./01.12345)6'7809:;.5)6'784 8!!&()*++&1.)E,#&-./01.123<=1+45)6'7809".5)6'784 8!!      !" #$%!&'(!!)!*!+&,-%-./+& 0###12&0*% %( &34567689:9;62%(&*!' ,!<=&#,#%>'0#?&0*%&*!"<=&#@,#=)#<=*!!)*% #A0&'%! )&B*  &!%* CD<CDE #F! &!% *  &!+=./)G./)-./-1./#E*%*'"(( !&% *%!&%0%0*0#HIJKLMNOPQRPMSTOUPVNLWUONTMXVHIJIYZQ[\WNPS[\UONTMXV#-##A<A, (!('0! %]A<A,D?Ê _D* !A<A,D?Ê !!0%̀]a''#)b1_#F00)%00*b>@>#!'%!00>@>#!'̀#F'0%c,de,D$$ ]1>1#>#1@!'f!00B>1#>#1@!'_*#2%!!&!(',+!(']A"_%00*!%  %̀&+  *(+*gGh#c& 0%0)%0 **%h]*i"_?0,?0]d%_0+&&#D' +(̀*"a''+&&]'e2i/ =#g)GhA<A)h0(!)#h+' %+*%*%@h]"i"_j,'! %+&-'b@./+&0%0#a00*-@'?]̂D= * (iÊ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ice-cold acetone, the pellet was dried for 10 min at room temperature, dissolved in 2x Laemmli buffer with 5 % (v/v) B-mercaptoethanol and heated at 95 °C for 5 min. 2.3.1.6 Determination of protein concentration For determination of protein concentration, duplicates of 3 and 6 w1 of protein solution were supplemented with 1 ml of 1:5 diluted Bradford Reagent (Bio-Rad). After incubation at room temperature for 10 min, the absorbance at 595 was measured and protein concentration determined by a calibration curve derived from absorbances of protein solution of known concentrations (0; 1,5; 3; 6; 12; 24 pg/ml) of bovine IgG standard. 2.3.1.7 Autoradiography For visualization of radioactively labeled proteins on nitrocellulose membrane, the membranes were dried under red light and exposed to X-ray films (Kodak Bio Max MM). After an appropriate time of exposure, protected from light by special Kodak film cassettes, the films were developed in a Gevamatic 60 developing machine (AGFA- Gevart). The exposure time was adapted to the signal intensities, and ranged from one day to several weeks. 2.3.1.8 Detection and quantification of ECL and radioactive signals on films Signals on sensitive films were detected and scanned with an ImageScanner (GE Healthcare) and quantified using the accompanying Master 1D Elite software (GE Healthcare). 2.3.2 Preparation of proteins 2.3.2.1 Overexpression of recombinantly expressed Ymel-AAA-Hise and MBP-Imp1 The AAA domain (amino acid residues 250 - 525) of S. cerevisiae Yme1 coupled to C-terminal His6-tag was expressed in the BL21 (DE3) E. coli strain from the pET28a+ expression vector (Novagen). Similarly, N-terminally MBP-tagged Imp] was as well expressed in the BL21 (DE3) E. coli strain from the pMAL-cRI vector (New England Biolabs). Overnight cultures of transformed BL21 (DE3) were used to inoculate 4 1 main MATERIALS AND METHODS 2.4 Cell biology methods 2.4.1 NaOH cell disruption (Kushnirov, 2000) 50 ml pre-culture was grown over night, diluted to an OD¢oo 0.2 in the morning and grown for 2 cell divisions until an OD¢oo of 0.6. 2 OD¢oo of culture were harvested by centrifugation at 14000 rpm (table top Eppendorf centrifuge) and room temperature for 5 min. The cell pellet was resuspended in 200 p11 0.1 M NaOH and incubated for 5 min at room temperature. Disrupted cells were pelleted by centrifugation at 14000 rpm (12154- H rotor/ Sigma) at room temperature for 5 min, resuspended in 50 pl 2x Laemmli with 5 % (v/v) B-mercaptoethanol per 1 OD¢oo and 0.5 - 1 OD¢oo were loaded per lane onto appropriate SDS-PA gels. 2.4.2 “Rédel’s” cell disruption (Horvath and Riezman, 1994) A second method for disruption of yeast cell is ‘Rédel’s’ cell lysis. Yeast cells were grown and harvested as described for NaOH lysis. The cell pellet was resuspended in 50 pl ‘Rédel’s’ mix (1.85 M NaOH, 7.4 % B-mercaptoethanol, 20 mM PMSF), vigorously mixed on the vortexer (VF2/ Janke und Kunkel) and incubated for 15 min on ice. Proteins were precipitated with TCA as described under 2.3.1.5 and analysed by SDS-PAGE, western blot and immuno-staining. 2.4.3 “Fast Mitoprep” Yeast strains were grown overnight, diluted in the morning and grown to OD¢o0 of 0.6 - 0.8. and 5 - 10 ODgo0 were collected in 15 ml reaction tubes and pelleted by centrifugation at 3000 rpm (SX4250 rotor/ Beckman X-22R Benchtop Falcon Centrifuge) for 5 minutes at room temperature. The pellet was resuspended in 300 pl of 0.6 M sorbitol, 20 mM Hepes, pH 7.4, 80 mM KCl, 2 mM PMSF, transferred to a 1.5 ml reaction tube and 200 ul cold glass beads (diameter: 0.5 mm) were added. Cells were lysed by four-times mixing steps with a vortexer (VF2/Janke und Kunkel) for 30 sec. Samples were placed on ice in between. The supernatant after centrifugation at 1000 xg and 4 °C for 3 min was transferred to a fresh reaction tube and spun down a second time for 10 min at 14000 rpm (12154-H rotor/ Sigma) and 4 °C. The supernatant of this spin was transferred to a new reaction tube and proteins were precipitated with the TCA method (see 2.3.1.5). Pellet fractions were directly resuspended in 20 pl 2x Laemmli      !"#!$#%&'"$!!$!$("%! #$)"*$$+ ,#*#% !$( (# #$(!%! #$*)*" %,)*- (#'.'/012)!$*(#$# %%#*!$,*$%#!$(!$!%,3(,$#*!$$+!$#(*!+!$*"#$*#$*!$((*# #$(!%!$( ,#*#% !4"#$*!* #$#%*&5676789:;<:=>?@A?BC#((!.!DEFGH)IJKLM#%!+* !%# #$(!%*#%!#$),!* %*+#$#LN(!,*#$# %!#$#!$ %LK%#!$O.PQQ# !&Q&I$$$+&R %* #$*!$%,4"$%#+! 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E 34!!!  &'33")#)/).1)8   3E 3 3 /I)#M!!  *//)#M!  3+!! )8     3%>3 !3  2B'>J7:F-<>7%   >J7:F-<>7 ! !!    ! 32B'>J7:F-<>7)XYZYX[\]̂_̀àbc_̀d̂daceef]ec_̀̂egfd_]̀̂ hijk[lmnĉ\ochhhg]b_fdo]_fj-> !p87H//qrsRt  %&9uK."0.Iv+>9>9w7*."07.H.Iv+?+1)x3  3 *x<8! 4#).6C!uyB !344%)< 3 3> !qrsRt % *! %*!3   )2   3!* 3> !  3   3  3qrsRt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̂@D) _ *IEG@ #"#̂@ ZI_ \̀ D MATERIALS AND METHODS Horizontal rotary shaker made in house PCR cycler “Mastercycler gradient” Eppendorf Pipettes, 10 pl - 5000 pl Gilson Sonifier 250 Branson Spectrophotometer Nanodrop 2000c Peqlab Thermomixer “comfort” Eppendorf 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̂5_CEV<VHPI&)̂5_CEV<VHP̀aVJI"&"0)8!"" !$"&" !#'"!)$!$7)0'!.%0,!49:;E)!*I0'*" !$0")#-E76+!I4)!.%!,"#L!652 RESULTS IMS-DHFR_ matrix-DHFR log__stat__ log __ stat - toe toe te + galactose = » DHFR =. - ee = Yme1 ee ee ee Tim50 Sean ent Figure 7. Optimization of model substrate expression Two sets of yeast cells harboring IMS- or matrix-DHFR™’ in pYES2 plasmid were grown in parallel and expression was induced in the logarithmic (log) phase (OD¢09 0.6) in one set and in the stationary (OD¢00 0.9) phase (stat) in the other set by activation of GAL promoter. Cells were harvested after two hours induction, disrupted with NaOH and expression levels were analyzed by immuno-staining with antibodies for indicated proteins. p, precursor, i, intermediate and m, mature forms of the DHFR constructs. 3.1.3 Expression of model substrate in S. cerevisiae Expression of the four model substrates was induced by activation of the GAL promoter and monitored over 120 minutes. Samples of the cultures were taken at different time points and total cell extracts were analyzed by SDS-PAGE, western blot and immuno-staining. The expression levels of the model substrates increased during time after induction (Fig. 8 A-D). The expression levels of the wild-type model substrates were slightly higher than the expression levels of the mutant model substrates (Fig 8 A, D). Moreover, the expression levels of matrix-DHFR were generally higher than in the intermembrane space (Fig. 8 C, D). In conclusion, these findings confirm that the model substrates can be stably expressed upon activation of the promoter and that the expression increases with time after activation. The lower expression levels of the folding incapable mutant DHFR constructs may be caused by their increased degradation. The lower expression levels in the intermembrane space could be attributed to a higher turnover of the overexpressed model substrates in this compartment. The volumetric capacity of the intermembrane space is low and therefore, this increased turnover of the overexpressed model substrates could prevent their aggregation. Unrelated yeast proteins of the cytosol (hexokinase) and the inner membrane (Tim17) were analyzed as controls. Neither of them                                  !  "#$#%&'()*)+,-)./.*-0'1-',231-,-'4'5'41.*'/2.6'/.718(.-')/1)/+'41'98('11)/6-0':.2'417;1-(,-'1<                =     > =  ?        >                     @          =      A             BC D      BEF D     BCG!D   BH D            B?=IDC   >    =       =   =      J)67('K#L)/'-)+1.*:.2'417;1-(,-''98('11)./M          =  =      N      E       OP Q      >   RHSTU?OBVWD  TU?OBETD       @ S  =            RHSTU?O                 !"#!$%&'()&)$"*+' )%( #$"&)',$-'.!*$ ,!/%- !$$012113412567$ #)8&),)$")!/8&! )$',)90:97/!&;3.$!$")2<'.8=),()&)'$'=>?)%+><@<A:BCDE(), )&$+=! '$%..*$!A, '$$-*,$-'$ +!%),'-'$, @FGH'$% #)$%"' )%. !"#!$%&'=.'&I)&8&! )$,0J!.K1E!* )&.).+&'$)LJ.31E$ )&.).+&'$),8'")LF)85E.' &M72E$ )&.)%' )'$%.E.' *&)/!&.!/N<A@FGH"!$, &*" ,2O!"'=?' !$!/(=%A >8)0BEP7'$%.* '$ 0QE@7"!$, &*" ,2       ! "# $  $ # $%&'()*+,*)'*+)-*.)(/01234+56'*78(9,:;<**.,4(=.)(;8*:5+,)+<:*/'+>-*.)(/'(9>4:;?@A0+BC+9-*DEFGHIJKKLMNO.('5*')(5*)*'-8.*)4*/(:58.>,)+)*(/PQRPRS*T6'*,,*5-(5*:,9<,)'+)*,I)4*8',)+<8:8);=+,)*,)*596(.6'()*+,*)'*+)-*.)N2(')48,69'6(,*I8,(:+)*5-8)(U4(.5'8+/'(-U*:,*T6'*,,8.>)4*-(5*:,9<,)'+)*,=*'*,(:9<8:8V*5+.5)'*+)*5=8)46'()*8.+,*WNX4*=8:5Y);6*-(5*:,9<,)'+)*,=*'*6+'):;5*>'+5*596(.+558)8(.(/)4*6'()*+,*+.5+6'()*+,*Y'*,8,)+.)/'+>-*.)(/'(9>4:;?@A0+=+,>*.*'+)*5B28>NJ?I:*/)6+.*:I:+.*?MNO.U(.)'+,)I)4*-9)+.)7*',8(.,(/)4*-(5*:,9<,)'+)*,=*'**.)8'*:;5*>'+5*5B28>NJ?I'8>4)6+.*:I:+.*?MBZ*,)=*<*'+.5[U4+)VIJKLLMNO.6'*,*.U*(/)4*0123,9<,)'+)*+.+:(>-*)4()'*T+)*I)4*=8:5Y);6*-(5*:,9<,)'+)*,=*'*,)+<8:8V*5*7*./9')4*'I+.5)49,)4*:*7*:,(/)4*,)+<:*/'+>-*.)8.U'*+,*5B28>NJ?I:*/)6+.*:I:+.*\MNX48,/8.58.>U+.<**T6:+8.*5<;<8.58.>(/-*)4()'*T+)*)(=8:5Y);6*0123I=48U48.)9'.:*+5,)(/9')4*',)+<8:8V+)8(.(/)4*.+)87*/(:5N]*8)4*'(/)4*-9)+.)/('-,=+,,)+<8:8V*5<;-*)4()'*T+)*B28>NJ?I'8>4)6+.*:I:+.*\M<*U+9,*)4*,9<,)'+)*+.+:(>U+..()<8.5)(9./(:5*50123NO.U(.U:9,8(.I)4*,*'*,9:),,4(=)4+))4*=8:5Y);6*0123U(.,)'9U),+,,9-*)4*8'.+)87*/(:58.)4*-8)(U4(.5'8+:8.)*'-*-<'+.*,6+U*+.58.)4*-+)'8TN ̂ ̂  # $  $%O,(:+)*5-8)(U4(.5'8+=*'*,(:9<8:8V*5=8)4X'8)(._YJ̀ ̀+.58.U9<+)*5=8)46'()*8.+,*WB&WM/('?̀-8.+)̀ab8.)4*6'*,*.U*+.5+<,*.U*(/-*)4()'*T+)*Bc)TMN[+-6:*,=*'*+.+:;V*5<;[0[Y&dCe+.58--9.(Y,)+8.8.>=8)4+.)8<(58*,+>+8.,)0123N8I8.)*'-*58+)*+.5-I-+)9'*/('-(/Oc[Y0123N,/I,)+<:*/'+>-*.)96(.6'()*+,*58>*,)8(.N RESULTS 3.2.2 Requirements for folding of DHFR in the IMS and matrix Next, it was asked if nucleotides and heat stress have an effect on the folding state of the DHFR constructs. To this end, one set of isolated mitochondria from cells expressing the model substrates was depleted of nucleotides. The ATP levels of a second set of these mitochondria were kept high. Both sets were subjected to a short heat shock at 42 °C for 3 min. One set of samples was kept at 25 °C as a control. After solubilization of the mitochondria with Triton X-100, pellet and supernatant fractions, representing aggregated and soluble proteins, were separated by centrifugation. At 25 °C, IMS-DHFR™” was found in the soluble fraction in the presence and absence of ATP (Fig. 13 A, lanes 1-4). However, upon heat shock, the mature (m) form of IMS-DHFR™' aggregated in an ATP- dependent manner (Fig. 13 A, lanes 5-8). In the presence of ATP, 14 % of IMS-DHFR™* aggregated (Fig. 13 A, lane 7). In the absence of ATP, aggregation increased to 89 % (Fig. 13 A, lane 5). The intermediate (i) form of IMS-DHFR™’, which is not yet cleaved by inner membrane peptidase and is thus still anchored to the inner membrane, aggregated almost completely upon heat shock (99 %) (Fig. 13 A, lanes 5+7). Matrix-DHFR™’ also aggregated in an ATP-dependent manner upon heat shock (Fig. 13 B, lanes 5-7). However, only a much smaller proportion (1 % or 3 %) than in the intermembrane space aggregated in the matrix upon heat shock (Fig. 13 B, lanes 5+7). This could be attributed to a higher capacity of the protein quality control systems of the matrix than of the ones in the intermembrane space. A B IMS-DHFRWT matrix-DHFRYT 25°C 42°C 25°C 42°C ‘ + + - + 5 + ATP PSPSPSPS PSPSPSPS i 7 = a ee OF i 2 8 3 7 9 191 % ND 100 ND 100 3 97 1 99 % m1 99 1 99 99 1 14:96 % —-—-_ = — Se we a SS timso —--<-+4-— a Hep Figure 13. Aggregation of wild-type DHFR constructs in IMS and matrix Isolated mitochondria were incubated under the indicated conditions, solubilized with Triton X-100, and soluble (S) and aggregate (P, pellet) fractions were separated by centrifugation and analyzed by SDS-PAGE and immuno-staining for indicated proteins. Singals of wild-type IMS-DHFR (A) and matrix-DHFR (B) were quantified in supernatant and pellet fractions and expressed as percentages of total protein. ND, not detectable. i, intermediate and m, mature form of IMS-DHFR. RESULTS 3.3.2 Confirmation of Ni-NTA pulldown by western blot and immuno-staining The results of the mass spectrometric analysis were confirmed with an unrelated method. For this purpose, Ni-NTA pulldown was performed as described above. This time, mitochondria were pretreated with ATP-depleting system, ATP-regenerating system or ADP prior to solubilization. Samples of the mitochondrial lysates (total) and the eluates (bound) were analyzed by SDS-PAGE, western blot and immuno-staining. Ymel was specifically co-isolated with IMS-DHFR™"-His (Fig. 16, lanes 11, 14), but not with matrix-DHFR™"-His (Fig. 16, lanes 12, 15, 18). Notably, Ymel was only co-isolated in the absence of nucleotides or in the presence of ADP (Fig. 16, ‘ID’ in ‘-/ADP-bound’). In the presence of ATP, Ymel was not co-isolated (Fig. 16, ‘ID’ in ‘ATP/bound’). These findings agree with the typical behaviour of chaperone-substrate interactions. The interaction is typically stabilized by ADP or in the absence of nucleotides, as the substrate total bound - ADP ATP - ADP ATP - ID mD - IDmD - IDmD - ID mD - IDmD - ID mD DHFR Yme1 Ssc1 Hsp60 Tim50 Tom40 Figure 16. Identification of potential folding helpers of IMS-DHFR Isolated mitochondria were solubilized with digitonin-containing buffer in the absence of nucleotides or in the presence of ATP or ADP. 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'!3')%< 8= ,8= -8>( "%!%!% !?! ! % %@ - + @ ,06    6  ) %1!%A "%!%") %$ ! )$% $"%1%A %* % "!%!'%!%")%,1 6!,1 ! ) %1!%!4)B - +6+,#7C6    3 %!"$ %!%% ( %* % $ %'#$5A "%!%")%0+% %0 0+#7C)!%$"%1 + 8 +6+,2C + ! 1  ! 1!$% !%#7C!%!4)2$5+A4"4%$ %"'! ' !A"! )DEFGHI' / 8 6 ,    =  = 7C 7C!%A$"%1&J' !03 A %* %  !" $"')!'%%!%!%A"'!  A"'!8C# 4"4%!!"!$! $) RESULTS 3.6.2 Endogenous levels of Ymel substrates in Ayme! strain The findings from the SILAC screen were confirmed by an independent method. For this purpose, the steady state levels of candidate proteins from the SILAC screen for which antibodies were available were determined in the Ayme/ and wild-type strains. The endogenous levels of Erv1, Mcs10, and Mcs27 were approximately two-fold higher in the absence of Ymel (Fig. 22, left panel). This is remniscent of the behavior of DHFR in the absence of Ymel, and suggests that these proteins are also proteolytically turned over by Ymel. The steady-state levels of the other substrate candidates from the SILAC screen, Mcs19, Fejl, Phb2, Gut2, and Dld1, were indistinguishable between wild-type and deletion strains (Fig. 22, left and middle panel), indicating that these proteins are not predominantly degraded by Ymel. The steady-state levels of a large number of other mitochondrial proteins residing in the four mitochondrial subcompartments, outer membrane, intermembrane space, inner membrane and matrix, were tested and none of them was affected in the absence of Ymel (Fig. 22, middle and right panel). _WT _Aymet _WT _Aymet _WT_ Ayme1 ——— J — — —— ~ Wg protein eS coos ete [SS vcsz7 I ona SSS Toms BES cuz BSSS timo SSS ssc1 Figure 22. Steady state levels of endogenous proteins aggregated in Ayme1 Endogenous levels of candidates from SILAC were analyzed in wild-type and Aymel mitochondria. Isolated mitochondria (5 and 15g) were analyzed by SDS-PAGE, western blot, and immuno-staining with antibodies against indicated proteins. WT, wild-type. RESULTS 3.6.3 Aggregation of endogenous Ymel substrates in mitochondria of Aymel strain In order to confirm the data form the SILAC screen, the aggregation assay was repeated and aggregate and soluble fractions were analyzed by SDS-PAGE, western blot and immuno-staining with antibodies against the substrate candidates from the SILAC screen. Importantly, Ervl, Phb2, Gut2, Mcs19 and Dld1 were indeed found in the aggregate fraction of Ayme/ but not of wild-type mitochondria (Fig. 23, left panel, lanes 1+3). The aggregation propensity of these proteins was even increased by a short heat shock at 42 °C for 3 min. Interestingly, Mcs27 and Fej1, two components of the recently identified mitochondrial contact site complex MICOS/MINOS/MitOS (Harner et al., 2011, Hoppins et al., 2011, von der Malsburg et al., 2011) aggregated in the absence of 25°C 42°C 25°C 42°C WT Ayme? WT Ayme7 “WT ayme? WT aymet PSPSPS PS. PSPSPSPS ND 100 ND ND ND 100 ND ND % ND 100 1 99 ND 100 2 98 % a er ND 100 8 92 1 99 13 87 A ND 100 ND 100 ND 100 ND 100 % 41 99 8 #92 20 80 55 45 % ND 100 ND 100 ND 100 ND 100 % 1 99 4 9 3 97 12 88 a % ND 100 5 95 1 99 6 94 % ND 100 ND 100 ND 100 ND 100 % 41 99 2 98 1 99 6 94 7 ND 100 ND 100 ND 100 ND 100 % ND 100 ND 100 ND 100 7 93 % ND 100 ND 100 ND 100 ND 100 % ND 100 ND 100 ND 100 ND 100 % % Figure 23. Aggregation of endogenous substrates in Aymel Mitochondria were pre-incubated for three minutes at 25 or 42 °C, solubilized with Triton X- 100-containing buffer and soluble, S and aggregate, P (pellet) fractions separated by centrifugation. Samples were analyzed by SDS-PAGE followed by immuno-staining with the indicated antibodies. The DHFR signals were quantified in supernatant and pellet fractions and expressed as percentages of total protein. ND, not detectable. 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̂(3$!0/'!(1#()0&'$<A"6A"$)0_̀a%D(.13* -&**')%/+14!$%%05-/6,&*(3:2'3'H0"*:2b1!0!(cdc4efgh$)0,*!&)23(!$)0$)$3+H02+'//:)(4*!$')')%.(&')0'1$!0-&(!')*7i8",'304!+-7 Q R S  Z[\]5'!(1#()0&'$(.13* -&**')%/+14!$%%05-/6,&-&4')1:2$!0.(&!#&/'):!*$!>A(&F>BC"*(3:2'3'H0,'!#8&'!()j46̀ 4̀1()!$')')%2:..&$)0*(3:23"c$)0$%%&%$!"e<-3!D.&$1!'()*,&*-$&$!02+1)!&'.:%$!'()7c$/-3*,&$)$3+H02+cdc4efgh",*!&)23(!$)0'//:)(4*!$')')%,'!#!#')0'1$!0$)!'2(0'*7i8",'304!+-7 RESULTS 3.6.7 Co-isolation of endogenous substrates of Yme1 with His-tagged Ymel Finally, I wanted to determine if Ymel interacts directly with the newly identified endogenous substrates. To answer this question, isolated mitochondria from a strain harboring N-terminally His-tagged Ymel were pre-treated with ADP or ATP and solubilized with digitonin-containing buffer. His-tagged Ymel was captured with Ni- NTA agarose beads and isolated. Samples of the mitochondrial lysate (total), the supernatant after capturing (flow through) and the eluates were subjected to SDS-PAGE, western blot and immuno-staining. The membrane was stained with antibodies against the newly identified Ymel substrates. nN nN a? a Ss SF 6 ADP ATP TFLE TFLETFL ETFLE <a ae 123456789 10 11 12 Figure 27. Co-isolation of Gut2 with His-tagged Ymel Isolated mitochondria of cells expressing N-terminally His-tagged Ymel were solubilized with digitonin-containing buffer in the presence of ATP or ADP. Samples were incubated with Ni-NTA agarose beads and specifically bound proteins eluted with Laemmli buffer containing 500 mM imidazol. 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