Fase I: Visualizar

5.4.6  The  genetic  link  between  HSP12  and  HSP26  and  genes  associated  with  the   vacuoles.  

  As  mentioned  earlier,  SGA  analysis  of  HSP12  and  HSP26  identified  a  number   of  genes  that  are  associated  with  the  vacuole.  This  is  of  particular  interest  as  there   is   a   vast   amount   of   evidence   linking   the   vacuoles   with   ageing   and   DR   mediated  

Chapter  5.  An  unbiased  approach  to  identify  genetic  interactions  of  HSP12/HSP26    

lifespan  extension.  The  vacuole  is  often  described  as  the  yeast  counterpart  of  the   mammalian  lysosome,  and  serves  a  role  in  many  processes;  it  is  the  destination  for   cellular   elements   targeted   for   degradation   by   autophagic   processes,   it   acts   as   a   regulator  of  cellular  ion  homeostasis  and  as  a  storage  reservoir  for  nutrients,  metal   ions  and  other  metabolites  (Tsuchiyama  and  Kennedy,  2012).  There  is  evidence  to   suggest   that   the   vacuoles   are   important   in   yeast   longevity.   During   DR,   the   yeast   vacuoles   undergo   vacuolar   fusion   events   leading   to   the   presence   of   one   large   vacuole,   which   replaces   the   normal   4-­‐5   present   during   physiological   conditions   (Tsuchiyama  and  Kennedy,  2012).  Interestingly,  two  genes,  which  are  required  for   vacuolar   fusion,  ERG6  and  NYV1,   are   also   required   for   lifespan   extension   by   DR   (Gaber  et  al.,  1989,  Nichols  et  al.,  1997,  Tang  et  al.,  2008).  In  addition  a  set  of  7  OSH   (oxysterol   binding)   genes   are   also   required   for   DR   mediated   lifespan   extension   (Tsuchiyama   and   Kennedy,   2012).   Deletion   of   all   7   OSH   genes   leads   to   fragmentation   of   vacuoles,   whereas   overexpression   of  OSH6  has   been   shown   to   extend  yeast  RLS  (Gebre  et  al.,  2012,  Tsuchiyama  and  Kennedy,  2012).  Interestingly,   the  vacuolar  membranes  of  replicatively  old  yeast  cells  have  been  shown  to  become   disordered  (Gebre  et  al.,  2012,  Tang  et  al.,  2008).  It  may  be  that  Hsp12  is  required   to  stabilise  the  vacuolar  membranes  with  increasing  age,  in  keeping  with  its  known   role  as  a  lipid  chaperone  (Welker  et  al.,  2010,  Herbert  et  al.,  2012).  

  Analysis   of   the   genes   identified   as   being   associated   with   the   vacuoles   revealed   one   gene,  TRX2,   which   was   common   to   both  HSP12  and  HSP26.   TRX2  

(ThioRedoXin)   is   a   cytoplasmic   thioredoxin   isoenzyme,   part   of   the   thioredoxin   system  in  yeast  and  is  required  for  maintenance  of  redox  homeostasis  (Trotter  and   Grant,  2002).  In  addition  to  redox  homeostasis,  it  is  thought  that  TRX2  plays  a  role   in   many   cellular   processes,   two   of   which,   include   protein   folding   and   repair   of   oxidatively  damaged  proteins  (Grant,  2001).  TRX2  plays  a  huge  role  in  protection  of   the  cell  against  oxidative  stress  and  deletion  of  the  TRX2  gene  results  in  extreme   sensitivity   to   H2O2   (Kuge   and   Jones,   1994).   Both  HSP12  and  HSP26  play   a   crucial   role   in  

protein   homeostasis   and   protection   of   the   cell   against   stresses.   The   finding   that  

TRX2  also  overlaps  in  cellular  functions  is  of  high  significance.  During  exposure  to   oxidative   stress,  TRX2  may   interact   with  HSP12  and  HSP26  to   prevent   protein  

Chapter  5.  An  unbiased  approach  to  identify  genetic  interactions  of  HSP12/HSP26    

experiments  will  now  be  performed  to  re-­‐confirm  the  genetic  interaction  between  

TRX2  and  HSP12  and  TRX2  and  HSP26  in  multiple  yeast  backgrounds.  It  will  also  be   important   to   perform   in   vivo  protein   aggregation   assays   on   trx2/hsp12∆   and  

trx2/hsp26∆   double   mutants   to   determine   if   they   show   increased   protein   aggregation  in  comparison  to  single  mutants  and  wildtype.          

  Autophagy   occurs   in   the   lysosome/vacuole   and   functions   to   degrade   intracellular   components   and   recycle   building   blocks   during   DR   and   exposure   to   stresses   (Aris   et   al.,   2013,   Yen   and   Klionsky,   2008).   Exposure   to   stresses   causes   proteins   to   unfold   and   impairs   proper   folding   of   nascent   proteins.   Unfolded   proteins  are  either  refolded  by  Hsps  or  are  targeted  for  destruction  by  one  of  three   processes:   chaperone-­‐mediated   autophagy   (CMA)   endosomal   microautophagy   or   by  chaperone-­‐assisted  selected  autophagy  (Kaushik  and  Cuervo,  2012).    

  Interestingly,  autophagy  is  linked  to  DR  and  ageing.  Autophagy  is  required   for   chronological   lifespan   extension   in   yeast   by   DR   and   promotes   mitochondrial   respiration  proficiency  during  ageing  with  DR  conditions  (Yen  and  Klionsky,  2008).   SGA  analysis  of  HSP12  and  HSP26  identified  two  genes,  ATG2  and  ATG3,  which  play   a   role   in   autophagy   and   are   also   linked   with   response   to   starvation.   It   is   thought   that  induction  of  autophagy  in  response  to  DR  is  required  for  removal  of  damaged   proteins  to  maintain  proper  metabolism,  which  is  crucial  for  cell  survival  (Yen  and   Klionsky,  2008).  The  link  between  HSP12  and  HSP26  and  genes  associated  with  the   vacuole  suggests  that  these  proteins  play  a  role  in  autophagy  processes  including   mitophagy.   Since   the  hsp12/hsp26∆  double   mutant   has   a   reduced   RLS   it   may   be   that  deletion  of  both  these  proteins  leads  to  defects  in  autophagy,  mitophagy  or  an   abnormality  in  vacuolar  membranes,  which  has  deleterious  effects  on  the  cell.  An   alternative  explanation  may  be  that  deletion  of  HSP12  and  HSP26  leads  to  a  loss  of   pH-­‐dependent   amino   acid   storage   in   the   vacuolar   lumen.   The   latter,   has   been   implicated   in   a   recent   theory   linking   the   vacuoles   and   the   mitochondria   in   yeast   ageing  (Hughes  and  Gottschling,  2012).  In  this  theory,  altered  vacuolar  pH  is  linked   with  a  loss  of  pH-­‐dependent  amino  acid  storage  in  the  vacuolar  lumen  leading  to   mitochondrial  dysfunction  and  ageing  (Hughes  and  Gottschling,  2012).  The  vacuoles   and  mitochondria  play  a  huge  role  in  ageing  and  the  link  identified  by  SGA  analysis  

Chapter  5.  An  unbiased  approach  to  identify  genetic  interactions  of  HSP12/HSP26    

between   these   two   organelles   and  HSP12  and  HSP26,  may   pinpoint   the   longevity   roles  of  these  proteins  to  processes  specific  to  the  vacuoles  and  mitochondria.