Klei, Ida Johanna van der

Partido Comunista de España

Biblioteca Virtual de Prensa Histórica (Virtual Library of Historical Newspapers)

Mundo Obrero  : Boletín del Partido Comunista de España en Francia: Número 175  - 1949 junio 23

Mundo Obrero  : Boletín del Partido Comunista de España en Francia: Número 149  - 1948 diciembre 23

University of Groningen Research Database

  
 University of Groningen
Alcohol oxidase in Hansenula polymorpha
van der Klei, Ida
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1991
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Klei, I. J. V. D. (1991). Alcohol oxidase in Hansenula polymorpha: assembly, activation and inactivation of a
peroxisomal enzyme s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
A  l i m i t e d  n u m b e r  o f  y e a s t  s p e c i e s  i s  c a p a b l e  o f  u t i l i z i n g
methano l  as  the  so le  carbon and energy  source  fo r  g rowth .  In
t h e s e  y e a s t s ,  t h e  f i r s t  s t e p  i n  m e t h a n o l  m e t a b o l i s m  i s  c a t a l y z e d
b y  t h e  e n z f r m e  a l c o h o l  o x i d a s e  ( A O ) .  T h i s  e n z y m e  o x i d i z e s
methanof ,  us ing  oxygen as  e lec t ron  acceptor ,  in to  fo rmaldehyde
and hydrogen perox ide .  In  i t s  ac t i ve  fo rm AO cons is ts  o f  e igh t
ident ica l  subun i ts ,  each o f  wh ich  conta ins  one f l -av in  aden ine
d inuc leo t ide  (FAD)  molecu le  as  the  pros the t ic  g roup.  In  v ivo  th j .s
f o r m  i s  c o n f i n e d  t o  t h e  m a t r i x  o f  s p e c i a l  c e l l  o r g a n e l l e s ,  c a l I e d
perox isomes.  These organe l les  a re  mass ive ly  induced dur ing  growth
of  ce l l -s  on  methano l - .  Depend ing  on  the  growLh cond i t ions  the
mat r ix  i s  par t l y  o r  comple te ly  c rys ta l l ine  due to  c rys ta lJ - i za t ion
of  AO pro te in .  L ike  a l l  perox isomal  enzymes s tud ied  so  fa r ,  AO
- is  encoded by  a  nuc lear  g ,ene and syn thes . ized  on  f ree  po lysomes
i n  t h e  c y t o s o l - .  A f t e r  i t s  s y n t h e s i s ,  t h e  m o n o m e r i c  p r o t e i n  i s
t rans loca ted  in to  perox isomes,  where  assembly  in to  the  ac t ive
oc tamer ic  enzyme occurs .
The molecu la r  mechan isms invo l -ved in  assembly ,  ac t i va t ion  and
inac t iva t ion  o f  AO were  }a rge ly  unknown a t  the  ou tse t  o f  the
inves t iga t ions .  The wea l - th  o f  in fo rmat ion  on  AO o f  HansenuLa
po lymorpha,  wh ich  a lso  inc luded the  nuc l -eo t ide  sequence o f  the
g e n e ,  r e g u l a t i o n  o f  e x p r e s s i o n  a n d  k i n e t i c  p r o p e r t i e s ,  m a d e  A O
of  th is  o rgan ism a  su i tab le  cand ida te  fo r  s tud ies  on  the
m e c h a n i s m s  o f  A O  a s s e m b l y ,  a c t i v a t i o n  a n d  i n a c t i v a t i o n .
T n  f - h A n t p r  1  t h e  C u r r e n t  k n O W l  e d . ' e  o n  n r o n c r t i e S  O f  t h e
p e r o x i s o m a l  e n z y m e  A O  i s  r e v i e w e d ,  i n c l u d i n g  i t s  s y n t h e s i s ,
impor t  in to  perox isomes,  assembly  and c rys ta l l - i za t ion .
Chapter  2  descr ibes  exper iments  in  wh ich  the  s tage o f  FAD b ind ing
dur ing  AO assembl -y  was s tud ied ,  The exper iments  were  based on  the
f ind ing  tha t  in  v ivo  FAD d issoc ia tes  f rom AO dur ing  incubat - ion
of  ce l l s  w i th  KCN,  caus ing  compLete  inac t iva t ion  o f  the  oc tamer ic
enzyme.  No reac t iva t ion  o f  the  apo-oc tamer ic  enzyme was observed
af te r  remova l  o f  KCN and incubat ion  o f  the  ce ] fs  in  f resh
methano l - -conta in ing  med ia .  S imi la r  resu l - ts  were  ob ta j -ned when the
media  were  supp lemented w i th  excess  FAD.  These f ind ings  suggested
tha t  FAD b ind ing  most  p robab ly  occurs  to  the  rnonomer ic  fo rm pr io r
to  oc tamer iza t ion .  The recovery  o f  AO ac t iv i t y  in  the  ce l l s  was
fu1 Iy  accounted  fo r  by  syn thes is  o f  new AO pro te in .  Ev idence was
obta ined tha t  a  he terogene i ty  ex is ts  among the  perox isomes
n r F s e n t  i n  f h e  r : c l I .  n r o t e i n  i m n o f t  w a s  c o n f i n e d  t o  S m a l f  a n d
,  Y _ "
deve lop ing  organe l les ,  whereas  the  la rge  organe l les  conta in - ing
inac t ive  AO,  were  pre ferab ly  p ro teo l -y t i ca l l y  degraded.
Proper t ies  o f  AO when expressed in  baker 's  yeas t  (Saccharomyces
c e r e v i s i a e )  a r e  d e s c r i b e d  i n  c h a p t e r  3 .  W i l d  t y p e  b a k e r ' s  y e a s t
does  no t  con ta in  AO and is  no t  capab le  o f  u t i l i z ing  methano l  as
a  so le  carbon and energy  source .  In  baker 's  yeas t ,  t rans formed
with the AO gene of H.poJyrrnorpha, the AO protein expressed was
par t l y  impor ted  in to  perox isomes,  whereas  most  o f  the  pro te in
res ided in  the  cy toso l  and was present  in  la rge  pro te in
aggregates .  Assembly  o f  AO in to  ac t ive  enz f rme and b ind ing  o f  FAD
to  the  AO pro te in  was no t  observed in  the  t rans formed baker 's
v e a s t -  i r r c q n e c t i v e  o f  i t s  S u b c e l - f u l a r  l O c a l i z a t i o n .  T h e  S a m e
gene was expressed in  fus ion  produc ts  o f  p ro top fas ts  p repared
f rom the  t rans formed s t ra in  and w i l -d  type  I t .  poTymorpha ce1 ls ,
under condit ions in which the nuclear gene of H. polymorpha was
comple te ly  repressed,  In  these fus ion  produc ts  AO ac t iv i t y  was
detec teC,  wh ich  was cy tochemica l , l y  demonst ra t .ed  to  be  conf ined
to  perox isomes o f  H.  po lymorpha.  These observa t ions  suggested
tha t  FAD b ind ing  and assembly  a re  no t  spontaneous processes  bu t
r e q u i r e  o n e  o r  m o r e  " a s s e m b l y  f a c t o r s " ,  p r e s e n t  i n  p e r o x i s o m e s
of  H,  poJynorphat  bu t  absent  in  the  organe lJ -es  o f  S .  cerev is iae .
Perox isome-def ic ien t  (PER)  mutants  o f  H.  po lynorpha were  iso la ted
f rom a  co l l -ec t ion  o f  s t ra ins ,  wh j -ch  were  de f ic ien t  in  the
ut i l i za t - ion  o f  methano l - .  Two mutants  comple te ly  lacked
recogn izab le  perox isomal  s t ruc tu res .  Genet ic  ana lys is  revea led
tha t  the  de f ic iency  to  u t i l - i ze  methano l  and the  absence o f
perox isomes in  a  PER mutant  can be  due to  a  muta t ion  in  a  s ing le
d a n o  l a h a n t a r  4 \
' t .
In  a  PER mutant  h igh  express ion  o f  AO was ob ta ined dur ing  growth
of  ce f ls  in  g lucose- l - im i ted  cont inuous  cu l tu res  in  the .presence
o f  c h o L i n e  a s  t h e  s o l e  n i t r o g e n  s o u r c e  ( c h a p t e r  5 ) . B y  ( i m m u n o - )
cy tochemis t ry  i t  was  shown tha t  AO was loca l i zed  in  the  cy toso l
l - o q
a n d  t h e  n u c L e u s .  A t  h i g h  e x p r e s s i o n  r a t e s ,  l a r g e  c r y s t a l l o i d s
were  fo rmed;  genera l - Iy  one cy toso l i c  c rys ta l lo id  was observed per
ce l1 ,  occas iona l l y  they  were  a l -so  encountered  ins ide  the  nuc l -e i .
The molecu la r  a rch i tec tu re  o f  the  c rys ta l lo ids  was s imi l -a r  to
those descr ibed fo r  AO crys ta l lo ids  in  perox isomes o f  w i ld  type
ce l l s .  B iochemica l  ana lys is  reveafed  tha t  the  pro te in  assembled
in to  ac t ive  oc tamer ic  enzyme as  e f f i c ien t ly  as  in  w i ld  type
ce l l s ,  suggest ing  tha t  pu ta t i ve  "assembly  fac to rs"  a re  a l -so
l -oca ted  and ac t ive  in  the  cy toso l  o f  the  mutant .
Chapter  6  descr ibes  s tud ies  on  methano l -metabof ism in  a  PER
mutant .  In  g lucose- l - im i ted  cont inuous  cu l tu res  us ing  methano l  as
a  second subs t ra te  in  the  feed,  a l l  perox isomal  enzymes invo l ,ved
in  methano l -metabo l ism were  expressed and ac t ive  a t  comparab le
l -eve1s  in  PER and w i ld  type  ce l - l s .  S imi la r ly ,  the  cy toso l i c
enzfrmes of the dissini latory pathway, formaldehyde dehydrogenase
and fo rmate  dehydrogenase,  were  induced to  the  same ex ten t  in
bo th  s t ra ins .  The add i t ion  o f  low concent ra t ions  o f  methano l -  to
the  feed o f  g lucose- l im i ted  cont inuous  cu l tu res ,  resu l ted  in  a
minor  inc rease in  ce l l  y ie ld  o f  the  PER cu l tu res .  However ,  when
h igher  methano l  concent ra t ions  were  used,  the  ce l I  dens i t ies
gradua l ly  decreased and f ina l l -y  d ropped be low the  or ig ina l  va l -ue
o f  t h e  c u l t u r e  p r i o r  t o  t h e  a d d i t i o n  o f  m e t h a n o l .  T h i s  w a s  i n
cont ras t  to  the  resu l ts  ob ta ined w i th  w i ld  type  cu l tu res  in  wh ich
a l inear  inc rease in  b iomass w i th  inc reas ing  amounts  o f  methanof
w a s  o b s e r v e d .  T h e s e  f i n d i n g s  s u g g e s t e d  t h a t  i n  P E R  c e I I s  a t
enhanced methano l -  suppJ-y  ra tes  the  metabo l ism o f  methano l  may
requ i re  energy  and, /o r  causes  tox ic  de fec ts .  The enzyme ac t iv i t ies
o f  two enzymes,  ind i rec t l y  invo lved in  methano l -  metabo l ism,  were
s ign i f i can t ly  d i f fe ren t  in  PER and w i ld  type  ce l l s .  Fur thermore
res idua l  methano l  and fo rmaldehyde,  excre ted  by  the  ce l l s ,  were
detec ted  in  the  PER cu l tu res .  The absence o f  in tac t  percx isomes
r e s u l t e d  m o s t  p r o b a b l y  i n t o  t w o  m a j o r  d r a w b a c k s ,  n a m e l y  i )  t h e
genera t ion  o f  hydrogen perox ide  in  the  cy toso l  and i i  )  the
inab i l i t y  o f  the  ce l l s  to  cont ro l  fo rmaldehyde f luxes  over  the
d i s s i m i l a t o r y  a n d  a s s i m i f a t o r y  p a t h w a y s .  A s  a  r e s u 1 t  H 2 O ,  a n d
formaldehyde are  most  p robab ly  metabo l ized  j -n  PER ce l - l s  v ia
pathways which are energetical ly fess advantag'eous or even energy
l - o f
consuming, when compared to the normal pathways in wild type
c e l f s .
In  PER and w i ld  type  ce l l s  a fso  d i f fe ren t  mechan isms o f  AO
inac t iva t ion  were  observed (chapter  '7  )  .  f  n  w i ld  type  ce l - l -s
se lec t ive  inac t iva t ion  o f  AO occurs  a f te r  t rans fer  o f  ce l l s  to
g lucose-  o r  e thano l - -conta in ing  med ia ,  wh ich  invo lves  pro teo ly t i c
tu rnover  o f  who le  perox isomes,  inc lud ing  a l l  mat r i x  enzymes.  When
P E R  c e f f s ,  c o n t a i n i n g  l a r g e  c y t o s o l i c  A O  c r y s t a l l o i d s ,  w e r e
t r a n s f e r r e d  t o  g l u c o s e - e x c e s s  c o n d i t i o n s ,  t h e  s u b s e q u e n t  d e c r e a s e
i n  A O  a c t i v i t y  c o u l - d  f u 1 l y  b e  a c c o u n t e d  f o r  b y  d i l u t i o n  o f  t h e
enzyme due to  g rowth ;  p ro teoJ-y t i c  degradat ion  o f  AO was no t
observed.  S imi la r  resu l ts  were  ob ta ined w i th  a  second PER mutant ,
impa i red  in  the  impor t  o f  perox isomal  enzymes.  Th is  mutant  i s
charac ter ized  by  the  presence o f  smal l  perox isomes together  w i th
a  l a r g e  c y t o s o l i c  A O  c r y s t a l l o i d .  U p o n  t h e  s h i f t  o f  c e l l s  t o
g lucose excess  cond i t ions  par t  o f  the  smal -L  o rganeJ- les  p resent
in  these ce l - l -s  were  degraded by  mechan isms s imi la r  to  those
d e s c r i b e d  f o r  w i l " d  t y p e  c e I I s .  P r o t e o J - y t i c  d e g r a d a t i o n  o f
cy toso l i c  AO crys ta l lo ids  was no t  observed.  These f ind ings
suggested  tha t  p ro teo ly t i c  degradat ion  o f  AO is  dependent  on  i t s
I o c a l i z a t i o n  i n s i d e  p e r o x i s o m e s .
Trans fer  o f  ce . l -1s  to  methano l -excess  cond i t ions  resu l ted  in
rnod i f i ca t ion  inac t iva t ion  as  has  been descr ibed fo r  w i ld  type
ce l l -s ,  Mod i f i ca t ion  inac t iva t ion  was a lso  observed when PER ce l - fs
were  t rans fer red  to  e thano l -conta in ing  med ia ,  cond i t ions  wh ich
i n  w i l d  t y p e  c e l l - s  l e a d  t o  p r o t e o l y t i c  d e g r a d a t i o n  o f
p e r o x i s o m e s ,  i n c l u d i n g  t h e  m a t r i x  p r o t e i n s .  A c c o r d i n g l y ,
phosphory la t ion  /  dephosphory la t ion  as  a  poss ib le  mechan ism o f
A O  m o d i f i c a t i o n  w a s  s t u d i e d  ( c h a p t e r  8 ) .  A s  a  f i r s t  s t e p  t h e
presence o f  the  key  enzymes invo l -ved in  th is  mechan ism was
inves t iga ted .  From both  b iochemj -ca1 and cy tochemica l -  exper iments
ev idence was ob ta ined tha t  bo th  p ro te j -n  k inase and phospho-
hydro lase  ac t iv i t ies  a re  assoc ia ted  w i th  perox isomes o f  H,
poJymorpha,  However ,  us ing  e i ther  in  v ivo  or  in  v i t ro
exper iments ,  phosphory l -a t ion  o f  AO cou l -d  no t  unegu ivoca l l y  be
demonst ra ted .
