Molecular  biology  methods

2.4.1  Amplification  of  DNA  fragments  by  PCR  

DNA  fragments  were  amplified  by  PCR  using  KOD  Hot  Start  DNA  polymerase  (Novagen)   according   to   the   manufacturers   specifications   in   50  µl   scale   (for   standard   reactions).  

100  ng  of  yeast  genomic  DNA  or  10  ng  of  plasmid  DNA  were  used  as  template.  Cycling   conditions  were  adjusted  to  the  length  of  the  fragment  and  melting  temperature  of  the   primer  pair.  

2.4.2  Purification  of  plasmids  

Plasmids  were  purified  from  E.  coli  using  Wizard®  Plus  SV  Minipreps  DNA  Purification   System   (Promega),   according   to   the   manufacturers   specifications.   Following   the   purification,   concentration   of   the   nucleic   acid   solution   was   measured.   Plasmids   were   stored  in  dH2O  at  -­‐20  °C.  

2.4.3  Preparation  of  yeast  genomic  DNA  

Yeast  cells  were  inoculated  in  YPGal  and  grown  overnight.  After  determination  of  OD600,   2.5  OD600  were  isolated  by  centrifugation  (2  min,  20000  xg,  at  RT).  The  supernatant  was   discarded,  the  pellet  was  resuspended  in  150  µl  solution  A  (50  mM  Tris/HCl  (pH  7.4),   10  mM  EDTA,  0,3%  β-­‐mercaptoethanol,  0.5  mg/ml  zymolyase)  and  incubated  at  37  °C   for   1   h   on   a   thermomixer   (350   rpm).   After   addition   of   SDS   to   a   final   concentration   of   1%,   0.5   volumes   of   8   M   ammoniumacetate   were   added,   mixed   thoroughly   and   incubated   at   -­‐20  °C   for   15   minutes.   The   lysate   was   cleared   by   centrifugation   (14000  rpm,   15   min,   4  °C)   and   180  µl   of   the   supernatant   were   taken   and   DNA   was   precipitated  by  addition  of  120  µl  isopropanol.  After  centrifugation  (14000  rpm,  15  min,   4  °C)   the   pellet   was   washed   with   70%   EtOH   once   and   dried.   After   resuspension   in   appropriate  volume  of  TE  buffer,  the  yeast  genomic  DNA  was  stored  at  -­‐20  °C.  

2.4.4  DNA  electrophoresis  

DNA  fragments  were  separated  by  horizontal  agarose  gel  electrophoresis.  1%  agarose   gels   were   prepared   in   TAE   buffer   and   supplemented   with   ethidiumbromide.   DNA   samples   were   mixed   with   loading   dye   (4x   stock:   40   %   saccharose,   1%   OrangeG).  

Electrophoresis  was  performed  in  TAE  buffer  for  20-­‐30  min  at  100  V.  GeneRuler  DNA   Ladder  Mix  (Fermentas)  was  used  as  a  standard.  Separation  of  the  DNA  fragments  was   documented  using  a  UV-­‐transilluminator.  

2.4.5  Determination  of  nucleic  acid  concentrations  

DNA   and   RNA   concentrations   were   measured   with   NanoVue   spectrometer   (GE  Healthcare)   at   260   nm.   One   OD260   was   assumed   to   correspond   to   50  µg/ml   for   dsDNA  and  to  40  µg/ml  for  RNA.  

2.4.6  Sequencing  of  DNA  

Sequencing   of   DNA   was   performed   by   GATC   Biotech   (Konstanz).   Sequences   obtained   were  viewed  and  compared  using  GeniousPro  (Version  5.3.6,  Biomatters).  

2.4.7  Site-­‐directed  mutagenesis  of  plasmids  

Site-­‐directed   mutagenesis   was   performed   using   the   QuickChange®   Site-­‐Directed   Mutagenesis  Kit  (Stratagene/Agilent)  according  to  the  manufacturers  specifications.  To   mutate  a  single  amino  acid  in  COX15,  30  ng  of  COX15^WT,  cloned  into  pRS416  was  used  as   template.  Overlapping  primers  were  designed  with  the  desired  mutation  in  the  middle   of   each   primer   (forward:   5’-­‐   CAG   TTG   GTC   ATG   AGG   ACA   TGT   GCG   TAC   GTT   G,   CAT   replaced   by   ATG;   reverse:   5’-­‐   C   ACA   TGT   CCT   CAT   GAC   CAA   CTG   AAC   TGT   AAC,   ATG   replaced   by   CAT). Cycling   conditions:   initial   denaturation   (95  °C,   30   sec),   followed   by   20   cycles   of   denaturation   (95  °C,   30   sec),   annealing   (55  °C,   1   min)   and   amplification   (68  °C,  8  min).  To  digest  methylated,  non  mutated  parental  DNA  template,  the  reaction   was  treated  with  DpnI  at  37  °C  for  1  h.  The  mutated  plasmid  was  transformed  into  XL1-­‐

Blue   supercompetent   cells,   supplied   with   the   kit,   according   to   the   general   E.   coli   transformation  protocol  (see  section  2.4.9)  with  the  following  modifications:  cells  were   left  on  ice  for  30  min  prior  to  transformation  and  heat  shock  was  performed  for  45  sec   instead  of  2  min.  The  mutagenesis  was  verified  by  sequencing.

2.4.8  Chromosomal  deletion  and  tagging  of  yeast  genes  

Chromosomal   deletions   as   well   as   tagged   versions   of   yeast   genes   were   generated   by   introduction   of   the  klTRP1,  HIS3MX6   or  natNT2   cassettes   using   PCR   based   strategies   (Knop  et  al.  1999;  Janke  et  al.  2004).  Cox15^FLAG  was  generated  using  pYM2.1  vector  (D.  

Mick,   Rehling   lab).   Mss51^SF  was   generated   using   a   modified   pYM2.1   vector,   kindly   provided   by   J.   Melin   (Rehling   lab).   Briefly,   the   streptavidin-­‐FLAG   tag   from   pESG-­‐

IBA_168   (cut   with  XhoI  and  BglII)  was   ligated   into   pYM2.1,   cut   with  SalI  and  BglII   (Alkhaja  et  al.  2012).  

Yeast   strains   were   transformed   with   PCR-­‐amplified   integration-­‐cassettes   using   the   lithium  acetate  method,  as  described  in  section  2.4.10.  Integration  into  the  genome  was   confirmed  by  PCR  or  Western  blot  analysis  of  the  target  protein.  The  strains  generated   in  this  study  are  listed  in  table  5.  

2.4.9  Transformation  of  E.  coli  

100  µl   of   rubidium   chloride   (RuCl)   competent  E.   coli   cells   were   thawed   on   ice   and   incubated   with   50   ng   plasmid   for   15   min   on   ice.   The   reaction   was   subjected   to   heat   shock   at   42  °C   for   2   min   and   subsequently   cooled   down   on   ice   for   3   min.   1ml   of   LB   media,  pre-­‐warmed  to  37  °C,  was  added  and  bacteria  were  allowed  to  grow  for  1  h  at   37  °C  with  shaking.  The  transformed  cells  were  centrifuged  down  (10000  rpm,  5  min  at   RT),   resuspended   in   200  µl   LB   and   plated   onto   LB-­‐Amp   agar   plates.   Plates   were   incubated  at  37  °C  over  night  until  appearance  of  single  colonies.  

2.4.10  Transformation  of  S.  cerevisiae  

Yeast   cells   were   transformed   by   the   LiOAc   method   (Ito   et   al.   1983;   Schiestl   &   Gietz   1989;   Gietz   &   Schiestl   2007).   Cells   were   inoculated   on   the   day   before   transformation   from  a  fresh  agar  plate  into  2x  YPAD  and  grown  overnight.  On  the  next  day,  the  main   culture   was   inoculated   with   the   pre-­‐culture   to   an   OD600   of   0.5   and   allowed   to   double   twice.   At   OD600=   2,   the   yeast   cells   were   harvested   (4000   rpm,   3   min,   18  °C),   washed   once   with   0.5   volumes   of   sterile   water   and   once   with   0.5   volumes   of   transformation   buffer  (0.1  M  LiOAc,  0.1  mM  EDTA,  5  mM  Tris/HCl  pH  8.0).  The  cells  were  subsequently   resuspended   in   transformation   buffer   (1/50   volumes   of   the   original   culture)   and   aliquoted   to   100   µl   per   reaction.   To   each   reaction,   1/10   volume   of   carrier   DNA   (10  mg/ml   herring   sperm   DNA,   boiled   at   95  °C   for   10   min   prior   to   addition)   and   the   target   DNA   (1-­‐2  µg   PCR   product/   30   ng   plasmid   DNA)   was   added.   For   a   control   reaction,   the   target   DNA   was   replaced   by   the   same   volume   of   sterile   water.   After   addition  of  600  µl  40%  PEG-­‐4000  in  transformation  buffer,  the  mixture  was  incubated   at  30  °C  for  30  min.  Heat  shock  was  performed  at  42  °C  for  15  min  after  addition  of  68  µl   DMSO.  Cells  were  harvested  by  centrifugation  (2000  rpm,  2  min  at  RT),  resuspended  in   200  µl   1   M   sorbitol   and   plated   onto   appropriate   selective   media   plates   under   sterile   conditions.  Plates  were  incubated  at  30  °C  until  appearance  of  colonies  (usually  3  days),   then   single   colonies   were   picked   and   re-­‐streaked   onto   selective   media   plates.   All   solutions  used  for  this  procedure  were  either  autoclaved  or  filter-­‐sterilized.