Jpn.  J.  Protozool.  Vol.  43,  No.  1.    (2010)

5  Review

Tubulin  polymodifications  in  Tetrahymena    Jacek  GAERTIG    Department  of  Cellular  Biology,  University  of  Georgia,  Athens,  GA  30602,  USA  Ciliates   have   a   microtubule-­rich   cytoskeleton.  
Tetrahymena   thermophila   assembles   at   least   18  types   of   distinct   microtubules  that   are  located   in  cilia,  cell  cortex,  nuclei  and  cytoplasm  of  the  cell  body.  Specific  microtubules  differ  in  the  filament  length  and  curvature,  degree  of  bundling,  level  of  subunit    turnover    and    levels    of    microtubule-­interacting   proteins.   How   diverse   microtubules  assemble   and   function   is   a   largely   unanswered  question,   to   which   ciliate   studies   have   recently  offered   important   insights.   Commonly,   multiple  variants    of    the    microtubule    building    blocks,  dimers  of  D-­/E-­tubulin,  are  expressed  in  the  same  cell.   Often   tubulin   variants   are   spatially   segre-­gated,   and   therefore   could   be   involved   in   the  functional   adaptations   of   specific   microtubules.  
Tubulin   variants   arise   by:   1)   expression   of   dis-­tinct  tubulin  isotypes  (products  of  distinct  genes)  and   2)   production   of   tubulin   isoforms   by   post-­
translational  modifications.    

*Corresponding  author    Tel:  +1  706  542  3409      Fax:  +1  706  542  4271    E-­mail:  jgaertig@cb.uga.edu   Received:  13  August  2009    Tubulin  isotypes  in  Tetrahymena    Early  studies  suggested  that  T.  thermophila  con-­tains   only   single   isotypes   of   D-­   and   E-­tubulin  (Gaertig   et   al.,   1993;;   McGrath   et   al.,   1994).   This  view  has  been  revised  on  the  basis  of  the  sequence  of   the   macronuclear   genome   (Eisen   et   al.,   2006).  
Tetrahymena  has  only  a  single  gene,  ATU1,  encod-­ing   a   conventional   D-­tubulin,   Atu1p   (McGrath   et  al.,   1994)   and   two   genes   for   a   conventional   E-­tubulin  (BTU1  and  BTU2)  that  encode  exactly  the  same   protein,   Btu1/2p   (Gaertig   et   al.,   1993).   The  amino   acid   sequences   of   Atu1p   and   Btu1/2p   are  over   90%   identical   to   orthologs   in   most   eukaryo-­tes.   Not   surprisingly,   Tetrahymena   needs   conven-­tional   D-­   and   E-­tubulin   for   survival.   ATU1   is   es-­sential   (Hai   et   al.,   1999).   While   the   expression  patterns  of  BTU1  and  BTU2  are  distinct  (Gu  et  al.,  1995),   Tetrahymena   needs   only   one   of   them   for  survival   (Xia   et   al.,   2000).   In   addition,   Tetrahy-­
mena   has   single   genes   encoding   highly   conserved  tubulins  that  are  associated  with  the  sites  of  micro-­
tubule  nucleation,  including  J-­  G-­,  K-­  and  H-­tubulin  (Eisen   et   al.,   2006;;   Shang   et   al.,   2002).   The   J-­tubulin   of   Tetrahymena   is   essential   and   required  for   duplication   and   maintenance   of   basal   bodies  (Shang  et  al.,  2002).  The  G,  K-­  and  H-­tubulin  have  6  原生動物学雑誌 第43巻 第1号 2010年  not   been   studied   in   Tetrahymena.   However,   these  tubulins  are  essential  in  Paramecium.  G-­tubulin,  as  shown   earlier   in   Chlamydomonas   (Dutcher   and  Trabuco,  1998),  is  required  for  the  assembly  of  the  C-­tubule   in   triplet   microtubules   of   the   basal   body  (Garreau  De  Loubresse  et  al.,  2001).  The  K-­  and  H-­tubulin  are  required  for  duplication  of  basal  bodies  (Dupuis-­Williams   et   al.,   2002;;   Ruiz   et   al.,   2000).  
Thus  ciliates  have  a  set  of  conserved  conventional  tubulins,   most   if      not   all   of   which   are   essential.  
However,   the   genome   of   Tetrahymena   also   con-­tains   predicted   genes   encoding   3   extremely   diver-­gent   D-­tubulin-­like   proteins   (ALT1   to   3)   and   9  equally   divergent   E-­tubulin-­like   proteins   (BLT1   to  6,  and  3  so  called  L-­tubulins)  that  appear  to  be  cili-­ate-­specific  (Aury  et  al.,  2006;;  Eisen  et  al.,  2006).  
Due  to  their  similarity  to  D-­  and  E-­tubulins,  the  D-­like   and   E-­like   tubulins   could   be   used   as   building  blocks   of   microtubules.   Thus,   contrary   to   the   ear-­lier  assumptions  (Gaertig  et  al.,  1993;;  McGrath  et  al.,   1994),   isotypic   diversity   could   play   a   major  role   in   ciliates.    In   vegetative   cells,   Atu1p   and  Btu1/2p,   are   incorporated   into   all   types   of   micro-­
tubules  (Thazhath  et  al.,  2004  and  our  unpublished  data).   It   is   likely   that   the   divergent   tubulins   (D-­like,   E-­like   and   L-­)   do   not   form   separate   micro-­
tubules,  but  instead  co-­assemble  with  conventional  tubulin.   Since   the   divergent   tubulin   isotypes   may  be   specific   to   ciliates,   these   proteins   could   be   in-­volved   in   assembly   of   types   of   microtubules   that  are   unique   to   ciliates.   Indeed,   in   Tetrahymena,   an  epitope   tagged   Blt1p,   is   not   targeted   to   axonemes  (K.    Clark,    M.    Gorovsky,    personal   communica-­tion),  organelles  that  contain  mostly  if  not  entirely  conventional  tubulin  dimers  (Gaertig  et  al.,  1995).  
The  divergent  D-­  and  E-­tubulins  could  co-­assemble  with   conventional   tubulin   in   microtubules   with  unusual   properties   such   as   the   cortical   bundles,  microtubules  that  form  during  amitosis  of  the  mac-­ronucleus  (Fujiu  and  Numata,  2000)  or  those  form-­
ing  the  contractile  vacuole  pore  (CVP).  CVP  con-­tains   a   small   ring   of   microtubules   twisted   into   a  right-­handed    helix    (Elliott    and    Bak,    1964;;  McKanna,  1973).  This  is  a  rare  example  of  micro-­
tubules   with   an   extreme   angle   of   curvature.   The  CVP   microtubules   may   require   an   unusual   degree  of   flexibility,   which   could   involve   unusual   bonds  between   tubulin   subunits.   In   the   future,   it   will   be  of  interest  to  determine  whether  divergent  tubulins  of   ciliates   have   amino   acid   substitutions   at   do-­mains   that   are   known   to   form   inter-­   and   intra-­dimer   surfaces   and   thus   affect   polymer   flexibility  (Nogales  et  al.,  1999;;  Nogales  et  al.,  1998).    
    Tubulin  isoforms  in  Tetrahymena    Tubulin  of  most  eukaryotes  undergoes  a  number  of    conserved    post-­translational    modifications  (PTMs).   Tetrahymena   tubulin   is   highly   heteroge-­neous    and    extensively    modified    post-­
translationally  (Gaertig  et  al.,  1995;;  Redeker  et  al.,  2005;;  Suprenant  et  al.,  1985).  The  most  conserved  and   well   studied   tubulin   PTMs   that   are   also   pre-­sent   in   ciliates   (Fig.   1)   include:   acetylation   of   D-­tubulin   (at   K40)   (Greer   et   al.,   1985),   proteolytic  removal   of   the   terminal   Y   residue   from   D-­tubulin  (detyrosination)   (Argarana   et   al.,   1978),   and   two  types   of   so-­called   polymodifications,   glutamyla-­tion   (Eddé   et   al.,   1990)   and   glycylation   (Redeker  et  al.,  1994)  (on  D-­  and  E-­tubulin).  With  exception  of   acetylation   at   K40   of   D-­tubulin   that   occurs   in-­side  the  microtubule  lumen  (Nogales  et  al.,  1999),  the   remaining   PTMs   occur   on   the   outside   surface  of   microtubules.   More   precisely,   detyrosination,  glutamylation   and   glycylation   occur   on   the   C-­terminal   tails   (CTTs)   of   tubulin,   flexible   domains  (Luchko  et  al.,  2008)  that  interact  with  motor  pro-­teins   and   other   types   of   microtubule-­associated  proteins   (MAPs)    (Roll-­Mecak   and    Vale,    2008;;  Skiniotis   et   al.,   2004;;   Wang   and   Sheetz,   2000).  
Tetrahymena  studies  showed  that  the  CTT  on  both  D-­  and  E-­tubulin  is  essential  (Duan  and  Gorovsky,  2002).  
Here   we   will   focus   on   PTMs   that   are   based   on  ligations  of  amino  acids  to  tubulin,  and  in  particu-­

Jpn.  J.  Protozool.  Vol.  43,  No.  1.    (2010)

7  Fig.  1.  Immunofluorescence  images  of  a  dividing  Tetrahymena  labeled  with  antibodies  that  recognize  either  the  tubulin  primary  sequence  (A)  or  various  post-­translational  form  of  tubulin.  The  following  antibodies  were  used:  (B)  GT335,  a  monoclonal  antibody  (mAb)  that  recognizes  mono-­  and  polyglutamyl  side  chain  (Wolff  et  al.,  1992),  (C)  ID5,  a  mAb  that  in  Tetrahymena  is  specific  to  polyglutamylation  (Rüdiger  et  al.,  1999;;  Wloga  et  al.,  2008),  (D)  6-­11  B-­1,  a  mAb  against  acetyl-­K40  on  D-­tubulin  (Piperno  and  Fuller,  1985),  (E),  TAP952,  a  mAb  against  monoglycylated  tubulin  (Bré  et  al.,  1998)  and  (F),  AXO49,  a  mAb  that  recognizes  polyglycy-­
lated  tubulin  (Bré  et  al.,  1998).  

lar  on  the  mechanism  and  function  of  polymodifi-­
cations   (glycylation   and   glutamylation).   Tubulin  undergoes    post-­translational    ligations    of    amino  acids  to  the  primary  polypeptide  that  utilize  protein  translation-­independent   mechanisms.   These   reac-­
tions   are   mediated   by   ATPase   enzymes   that   func-­
tion  as  amino  acid  ligases  (see  below).  Arce,  Arga-­
rana  and  colleagues  first  reported  that  D-­tubulin  is  modified   by   post-­translational   ligation   of   tyrosine  to  the  C-­terminal  glutamic  acid  (Arce  et  al.,  1975;;  Argarana   et   al.,   1977).   Tyrosination   is   a   reverse  reaction  for  a  post-­translational  modification  based  on  proteolytic  removal  of  the  genome-­encoded  C-­terminal  tyrosine  by  a  carboxypeptidase  (Argarana  et   al.,   1978;;   Hallak   et   al.,   1977).   Eddé   and   col-­leagues   discovered   that   murine   brain   tubulin   un-­dergoes  post-­translational  addition  of  one  or  multi-­
ple   glutamic   acids   (glutamylation)   (Eddé   et   al.,  1990).  Redeker  and  colleagues  showed  that  axone-­mal   tubulin   of   Paramecium   is   extensively   modi-­
fied  by  ligation  of  glycines    (glycylation)  (Redeker  et   al.,   1994).   Unlike   tubulin   tyrosination   that   is  based  on  a  standard  peptide  bond,  glycylation  and  glutamylation  involve  isopeptide  bonds  that  utilize  the  J-­carboxyl  group  in  the  side  chain  of  an  inter-­nal  glutamic  acid  of  CTT.  The  first  added  glycine  or  glutamate  can  be  extended  into  a  side  chain  by  standard   isopeptide   bonds   (Redeker   et   al.,   1991;;  Regnard  et  al.,  1998;;  Rogowski  et  al.,  2009;;  Wolff  et   al.,   1994).   To   some   extent,   polymodifications  resemble   PTMs   based   on   post-­translational   liga-­tion   of   peptides,   (e.g.   ubiquitination   or   neddyla-­tion),  except  that  the  unit  of  polymodifications  is  a  single   amino   acid.   Due   to   the   polymeric   nature,  glycylation    and    glutamylation    generate    a    large  number   of   tubulin   isoforms   as   a   consequence   of  utilization  of  multiple  modification  sites  (glutamic  acids   within   the   CTTs),   variable   length   of   side  8  原生動物学雑誌 第43巻 第1号 2010年  chains   and   the   fact   that   both   polymodifications  often  co-­exist  on  the  same  tubulin  subunits  in  vari-­ous    combinations    (reviewed    in    (Gaertig    and  Wloga,   2008).   It   came   as   a   surprise,   that   despite  major  differences  between  the  biochemical  mecha-­nisms   of   tyrosination   and  polymodifications,   all  3  PTMs   are   generated   by   structurally   related   en-­zymes  (see  below).    
    Tubulin  glutamylation    Tubulin   glutamylation   is   present   in   most   eu-­
karyotes   with   a   possible   exception   of   fungi.   The  phylogenic   patterns   suggest   that   this   PTM   co-­evolves    with    cilia    and    centrioles/basal    bodies  (Janke   et   al.,   2005).   While,   axonemes   and   centri-­
oles/basal   bodies   are   highly   enriched   in   glutamy-­
lated   tubulin,  typically   in   the  same   cell,   this   PTM  is  also  present  on  other  types  of  microtubules.  For  example,   mammalian   fibroblasts   have   high   levels  of   tubulin   glutamylation   in   primary   cilia   and   the  centrosome,   but   the   PTM   is   weakly   detectable   on  cytoplasmic  and  spindle  microtubules  (Bobinnec  et  al.,   1998;;   van   Dijk   et   al.,   2007).   Bré   and   col-­leagues  have  documented  the  distribution  of  gluta-­
mylated   tubulin   in   Paramecium   and   Tetrahymena  by    immunofluorescence    with    a    glutamylation-­
specific   antibody   (Bré   et   al.,   1994)   and   later   con-­firmed  by  mass  spectrometry  of  purified  tubulin  of  Tetrahymena  (Redeker  et  al.,  2005).  In  both  Para-­
mecium  and  Tetrahymena,  glutamylation  is  present  in  most  if  not  all  types  of  microtubules.  However,  the   extent   of   glutamylation   is   spatially   regulated.  
The   formation  of   the   glutamyl   side   chain   consists  of   two   distinct   steps:   1)   initiation   based   on   a  isopeptide    peptide    bond    that    involves    the    J -­carboxyl  group  of  the  glutamic  acid  in  the  primary  sequence   and   2)   elongation   based   on   a   standard  isopeptide   bond  (Redeker   et   al.,   1991;;   Regnard   et  al.,  1998;;  Wolff  et  al.,  1994).  A  number  of  mono-­  and   polyclonal   antibodies   have   been   developed  that  recognize  glutamyl  side  chains  made  of  a  spe-­cific  number  of  E  residues.  Perhaps  the  most  com-­plete   picture   of   glutamyl   side   chain   distribution  emerged   from   our   recent   studies   in   Tetrahymena  (Wloga  et  al.,  2008).  It  appears  that  every  type  of  a  microtubule  in  Tetrahymena  contains  at  least  some  subunits  that  have  glutamyl  side  chains.  However,  the  upper  limit  of  the  glutamyl  side  chain  is  micro-­
tubule    type-­specific.    Intracytoplasmic,    nuclear  microtubules  and  a  subset  of  cortical  microtubules  have      side   chains   limited   to   monoglutamylation.  
Side    chains    with    an    upper    limit    of    2    Es  (biglutamylation)   are   present   in   the   postoral   fiber  and  CVP.  Basal  bodies  and  cilia  are  the  only  loca-­
tions   containing   side   chains   composed   of   3   or  more   Es   (Wloga   et   al.,   2008).   However,   in   loca-­
tions  that  contain  the  longest  side  chains  there  is  a  whole   range   of   tubulin   subunits   having   from   0   to  20  Es  per  tubulin  (Redeker  et  al.,  2005).  Moreover,  the  distribution  of  glutamylated  subunits  within  the  microtubule   is   far   from   random.   Immunofluores-­cence   studies   in   several   ciliated   models   showed  that   in   the   axoneme,   the   density   of   glutamyl   side  chains   changes,    forming   an   increasing   gradient  from    the    tip    of    cilia    toward    the    basal    body  (Fouquet  et  al.,  1994;;  Huitorel  et  al.,  2002;;  Kann  et  al.,  1995;;  Lechtreck  and  Geimer,  2000).  Strikingly,  within  the  peripheral  doublet  microtubule,  most  of  glutamylation    is    located    in    the    B-­tubule  ((Lechtreck   and   Geimer,   2000;;   Multigner   et   al.,  1996)   and   our   unpublished   results   for   Tetrahy-­mena).  Thus,  enzymes  that  generate  glutamylation  (E-­ligases,   see   below)   may   be   selectively   binding  to  specific  microtubule  surfaces.    
One  straightforward  mechanism  that  could  con-­trol  the  side  chain  density  and  length  is  the  age  of  the   microtubule   polymer.   There   are   major   differ-­
ences   in   the   rate   of   subunit   exchange   among   spe-­cific    types    of    microtubules.    In    Tetrahymena,  ³SXOVH-­FKDVH´ W\SH H[SHULPHQWV ZLWK DQ HSLWRSH-­
tagged   tubulin   showed   that   cytoplasmic,   nuclear,  and   longitudinal   cortical   (LM)   microtubules   turn-­
over   rapidly,   while   basal   body,   transverse,   post-­
ciliary   and   axonemal   microtubules   turn   over   very  Jpn.  J.  Protozool.  Vol.  43,  No.  1.    (2010)

slowly  (Thazhath  et  al.,  2004).  The  types  of  micro-­
tubules   that   have   short   glutamyl   side   chains   are  mostly   the   dynamic   cell   body   microtubules   that  have   a   short   life   span.   Thus,   these   microtubules  may  not  exist  for  a  sufficient  time  to  acquire  long  glutamyl    side    chains.    On   the    other    side,    most  microtubules   that   exchange   subunits   very   slowly,  such  as  basal  bodies  and  cilia,  also  have  the  long-­
est   glutamyl   side   chains.   While   the   microtubule  age   could   play   some   role,   additional   mechanisms  are   needed   for   generation   the   precise   pattern   of  glutamylation,    including    restrictions    to    specific  microtubule    surfaces.    Furthermore,    in    Tetrahy-­
mena,  artificial  stabilization  of  dynamic  monoglu-­
tamylated  microtubules  by  paclitaxel  does  not  lead  to  accumulation  of  long  glutamyl  side  chains  (our  unpublished   data).   This   argues   that   the   length   of  glutamyl   side   chains   is   primarily   regulated   at   the  level  of  activity  of  tubulin  modifying  or  demodify-­
ing  enzymes.    
    Tubulin  glycylation      The  first  hint  of  the  existence  of  tubulin  glycyla-­tion   came   from   studies   that   utilized   antibodies  generated   against   axonemal   tubulin   of   Parame-­
cium    tetraurelia.    These    antibodies    recognized  ciliary   tubulin   in   a   variety   of   species   but   often  failed   to   cross-­react   with   non-­ciliary   cytoplasmic  microtubules   in   the   same   cells   (Adoutte   et   al.,  1985).    Mass    spectrometry    showed    that    these  axoneme-­tubulin   specific   antibodies   recognize   a  polyglycine   side   chain   attached   to   either   D-­   or   E-­tubulin.    While   generally,    tubulin    glycylation    is  found   only   in   cells   with   cilia   (Levilliers   et   al.,  1995),  in  ciliates,  this  PTM  is  also  present  on  non-­ciliary  microtubules  (Adoutte  et  al.,  1991;;  Iftode  et  al.,   2000;;   Thazhath   et   al.,   2002).   In   Tetrahymena  the   spatial   distribution   of   glycylation   resembles  that   of   glutamylation.   Thus,   the   long   side   glycyl  chains  are  present  in  cilia,  basal  bodies  and  cortical  microtubules,    while    cytoplasmic    and    nuclear  9  microtubules  have  side  chains  limited  to  monogly-­
cylation   (Thazhath   et   al.,   2004;;   Thazhath   et   al.,  2002;;  Xia  et  al.,  2000).  Mass  spectrometry  studies  on   ciliary   tubulin   detected   from   0-­42   G   residues  per   tubulin   (Redeker   et   al.,   2005).   However,   as   is  the  case  of  glutamylation,  within  the  organelle,  or  possibly   a   single   microtubule,   glycylation   is   dis-­tributed   non-­uniformly.   Within   the   axoneme   of  Tetrahymena,    both    mono-­    and    polyglycylation  could   not   be   detected   on   the   central   pair   micro-­
tubules,   but   were   abundant  on   the  peripheral  dou-­
blets   (Wloga   et   al.,   2009).   It   is   not   known   yet  whether,    like   glutamylation,   glycylation   is   also  confined   to   the   B-­tubule   of   outer   doublets   in   Tet-­rahymena,  but  this  is  the  case  of  axonemes  of  sea  urchin   sperm   (Multigner   et   al.,   1996).   Multiple  adjacent  glutamic  acids  in  the  tail  domains  of  both  D-­  and  E-­tubulin  are  used  for  glycylation,  based  on  mass    spectrometry    studies    in    Paramecium    and  mutational  studies  that  located  homologous  sites  in  Tetrahymena  (Vinh  et  al.,  1999;;  Xia  et  al.,  2000).    
    Significance   of   sites   of   polymodifications   on  tubulin    Our   laboratory   has   studied   the   significance   of  E  residues   on   CTTs   of   D-­   and  E-­tubulin   in   Tetra-­hymena   that   either   are   homologous   to   residues  known   to   serve   as   sites   of   polymodifications   in  other   organisms   or   are   adjacent   to   these   sites.  
Charge-­conserving    amino    acid    substitutions    of  CTT   (E   to   D)   were   used   to   assess   how   important  the  polymodification  sites  are  in  vivo.  These  stud-­ies   have   benefited   greatly   from   the   use   of   hetero-­karyon   strains   that   have   disruptions   of   ATU1   or  BTU1  and  BTU2  genes  in  the  micronucleus.  When  tubulin  heterokaryons  mate,  their  progeny  dies  but  could  be  rescued  by  introduction  of  a  correspond-­
ing  tubulin  gene  (Hai  et  al.,  1999;;  Xia  et  al.,  2000).  
The   rescue   method   was   used   to   rapidly   assess   the  significance  of  multiple  sites  of  polymodifications.  
Sites  of  polymodifications  on  the  Atu1p  D-­tubulin  10  原生動物学雑誌 第43巻 第1号 2010年  are   not   essential   (Wloga   et   al.,   2008;;   Xia   et   al.,  2000).   In   fact,   all   5   glutamic   acids   of   CTT   on   D-­tubulin  could  be  replaced  by  aspartates,  leading  to  complete   loss   of   polymodifications   from   the   D-­tubulin  subunit.  The  resulting  mutant  cells  (ATU1-­
5D)  are  viable  but  grow  more  slowly  (Wloga  et  al.,  2008).   The   tail   domain   of   Btu1/2p   E-­tubulin   con-­tains  five  Es  that  are  homologous  to  the  glycylated  residues  in  Paramecium  (Vinh  et  al.,  1999;;  Xia  et  al.,   2000).   Tetrahymena   can   tolerate   substitutions  of   1   or   2   specific   polymodifiable   Es;;   however,  triple  or  quadruple  mutations  are    lethal  or  deleteri-­ous  (Xia  et  al.,  2000).  Mutants  with  a  lethal  triple  site  mutation  develop  a  novel  phenotype:  undergo  3-­4  cell  cycles  without  completing  cytokinesis  and  IRUP  ³FHOO-­FKDLQV´  7KHVH  FRPSRXQG  FHOOV  FDQassemble  only  excessively  short  disorganized  cilia  that   lack   central   microtubules   and   have   defects   in  the   peripheral   doublet   microtubules   (Thazhath   et  al.,   2004;;   Thazhath   et   al.,   2002).   The   cytokinesis  arrest  in  the  lethal  triple  polymodification  site  mu-­tant  is  associated  with  lack  of  severing  of  LM  cor-­tical   bundles   (Thazhath   et   al.,   2004;;   Thazhath   et  al.,   2002).   It   is   likely   that   the   a   failure   to   sever  LMs  blocks   the   ingression   of  the   cleavage   furrow  (Thazhath  et  al.,  2004).  These  studies  indicate  that  Es   that   serve   as   polymodification   sites   are   essen-­tial.   The   limitation   of   these   studies   is   that   these  sites  could  potentially  be  used  by  both  glutamyla-­tion  and  glycylation  and  the  sites  used  by  glutamy-­
lation   have   not   been   mapped   for   Tetrahymena.  
Moreover,   it   is   not   clear   to   what   extent   the   ob-­served   phenotypes   of   mutations   on   E-­tubulin   are  loss  or  gain-­of-­function.  For  example,  substitutions  of  polymodification  sites  on  one  of  the  two  tubulin  subunits,  change  the  composition  of  polymodifica-­
tions   on   the   partner   (non-­mutated)   subunit.   In   a  viable   E-­tubulin   mutant   lacking   3   adjacent   poly-­
modification  sites,  there  is  an  increase  in  the  level  of   glycylation   and   decrease   in   the   level   of   gluta-­
mylation   on   the   non-­mutated   D-­tubulin   subunit  (Redeker   et   al.,   2005).   It   is   possible   that   some  modifying  enzymes,  in  the  absence  of  high  affinity  target  sites  on  one  tubulin  subunit,  modify  similar  sites   on   the   partner   subunit.   In   fact,   this   has   been  observed   for   some   tubulin   E-­ligases   (van   Dijk   et  al.,  2007).  It  is  not  known  whether  excessive  levels  of   polymodifications   on   the  partner   subunit  in  the  tubulin   polymodifications   site   mutants   are   com-­pensating   or   contribute   to   the   mutant   phenotype.  
To  determine  the  consequences  of  loss-­of-­function  of    polymodifications    sites,    future    studies    are  needed  to  mutate  the  glutamic  acids  on  both  D-­  and  E-­tubulin  in  a  single  strain  of  Tetrahymena.    
Importantly,  in  Tetrahymena,  the  phenotype  of  a  lethal   triple   mutation   in   the   polymodification   do-­main  on  E-­tubulin  is  almost  precisely  phenocopied  by   knockouts   of   genes   that   encode   subunits   of  katanin,  a  microtubule-­severing  protein  (Sharma  et  al.,   2007).   These   observations   open   a   possibility  that  katanin  and  polymodifications  act  in  the  same  pathway.  One  possibility  is  that  katanin  selectively  recognizes   tubulin  subunits   that   are   polymodified.  
Katanin   and   related   spastin   bind   to      the   tubulin  CTT   (McNally   and   Vale,   1993;;   Roll-­Mecak   and  Vale,   2008;;   White   et   al.,   2007).   An   antibody   that  binds  to  a  terminal  glutamic  acid  (to  either  a  dety-­rosinated  end  of  D-­tubulin  or  glutamyl  side  chain),  blocks   spastin-­mediated   microtubule   severing   ac-­tivity   in   vitro   (Roll-­Mecak   and   Vale,   2008).   A  deficiency  in  katanin  activity  could  lead  to  lack  of  severing   of   LM   microtubules   during   cytokinesis  and  this  could  explain  the  cytokinesis  arrest    in  the  triple   polymodification   site   E-­tubulin   mutants   of  Tetrahymena.  Indeed  a  GFP-­katanin  fusion  protein  localizes   to   LMs   in   Tetrahymena   (Sharma   et   al.,  2007).   It   is   more   difficult   to   explain   how   katanin  contributes   to   assembly   of   cilia.   It   appears   to   ka-­tanin  acts  inside  cilia,  possibly  on  the  outer  doublet  microtubules,   that   are   also   heavily   polymodified  (Dymek   et   al.,   2004;;   Sharma   et   al.,   2007).      It   re-­mains   to   be   determined   whether   in   the   context   of  ciliary   assembly,   katanin   acts   as   a   microtubule  severing   factor,   or   mediates   another   related   activ-­
ity  on  the  surface  of  microtubules.    
  Jpn.  J.  Protozool.  Vol.  43,  No.  1.    (2010)

The  enzymes  that  generate  tubulin  polymodifi-­
cations  (E-­  and  G-­ligases)    Regnard   and   colleagues   developed   an   in   vitro  assay   for   tubulin   glutamylation  and  partially   puri-­fied  a  tubulin  E-­ligase  activity  from  murine  brains  (Regnard  et  al.,  1998;;  Regnard  et  al.,  1999).  Strik-­
ingly,   one   of   the   proteins   in   the   complex   was  found   to   be   TTLL1,   a   protein   with   a   conserved  domain   related   to   a   tubulin   enzyme   previously  purified    by    Weber    laboratory,    tubulin    tyrosine  ligase   (Janke   et   al.,   2005).   TTL   is   responsible   for  ligation   of   tyrosine   to   the   terminal   glutamic   acid  that   is   exposed   by   detyrosination   (Schroder   et   al.,  1985).   While   TTL   (Y-­ligase)   is   a   reverse   PTM  enzyme   and   E-­ligase   is   a   forward   PTM   enzyme,  there   are   similarities   between   the   reactions   cata-­
lyzed   by   these   two   enzymes.   Namely,   both   en-­zymes   utilize   a   glutamic   acid   as   a   modification  site,  and  in  both  cases  the  target  residue  is  present  within   the   CTT   of   tubulin.   Subsequent   studies  showed  that  both  E-­ligase  and  G-­ligase  indeed  are  members   of   the   TTLL   superfamily.   TTLL1,   and  several   other   members   of   the   TTLL   superfamily  including  TTLL4,  TTLL6  and  TTLL9  are  catalytic  subunits  with  tubulin  E-­ligase  activity  (Ikegami  et  al.,  2006;;  Janke  et  al.,  2005;;  van  Dijk  et  al.,  2007;;  Wloga   et   al.,   2008).   Gene   knockouts   or   knock-­downs    of    mRNA    encoding    specific    TTLL    E-­ligases   or   associated   proteins   greatly   decrease   the  levels    of    tubulin    glutamylation    on    subsets    of  microtubules   in   diverse   models:   mouse,   zebrafish  and   Tetrahymena   (Ikegami   et   al.,   2007;;   Janke   et  al.,  2005;;  Pathak  et  al.,  2007;;  Wloga  et  al.,  2008).  
Furthermore,   in   one   case,   of   the   murine   TTLL7  enzyme   (that   belongs   to   the   TTLL6   clade)   it   was  possible   to   reconstitute   the   in   vitro   tubulin   gluta-­
mylation   reaction   using   a   recombinant   E-­ligase  (Ikegami    et    al.,    2006;;    Mukai    et    al.,    2009).  
(Ikegami  et  al.,  2006;;  Janke  et  al.,  2005;;  Pathak  et  al.,   2007.      The   clades   of   E-­ligases   are   fairly   well  conserved   across   eukaryotes,   indicating   that   sub-­classes   of   E-­ligases   exist   that   differ   in   some   con-­

11  served   enzymatic   properties.   It   appears   that   the  major   differences   among   the   E-­ligases   of   distinct  TTLL  classes  are  based  on  1)  the  preference  for  Į-­  or  ȕ-­tubulin  and  2)  ability  to  either  initiate  or  elon-­gate  the  side  chains.    As  an  example,  TTLL1  pro-­teins  (based  on  studies  in  Tetrahymena  and  mouse  studies)      have   an   E-­ligase   activity   that   prefers   Į-­tubulin   and   in   Tetrahymena   this   enzyme   has   a  strong   side   chain   initiating   activity   (Janke,   2005;;  van   Dijk   et   al.,   2007;;   Wloga   et   al.,   2008).   Dele-­
tions   of   a   TTLL1   type   enzyme   (Ttll1p)   and   a  closely   related   TTLL9   type   enzyme   (Ttll9p)   in  Tetrahymena   led   to   a   major   loss   of   glutamylation  in   the   basal   bodies   (Wloga   et   al.,   2008).   Double  knockout   cells   grow   more   slowly   and   have   fewer  ciliary   rows.   Basal   bodies   with   an   apparently   ma-­ture   morphology   that   failed   to   dock   at   the   plasma  membrane    were    observed    in    the    cell    body    of  TTLL1  and  TTLL9  double  knockout  strains.  A  GFP  fusion   of   Ttll1p   localized   to   basal   bodies   while  Ttll9p   was   found   in   both   basal   bodies   and   cilia.  
Both  of  these  enzymes  primarily  modify  D-­tubulin  (Wloga   et   al.,   2008).   Thus,   glutamylation   on   D-­tubulin,   while   clearly   not   essential,   could   be   im-­portant   in   regulation   of   the   fidelity   of   basal   body  maturation.   A   mutation   in  PGs1,   a   protein   associ-­
ated   with   TTLL1   E-­ligase   in   the   mouse   causes  severe   defects   in   assembly   of   sperm   axonemes  (Campbell   et   al.,   2002).      In   Tetrahymena,   knock-­outs   of   genes   encoding   of   multiple   paralogs   of  TTLL6  type  enzyme  led  to  shortening  of  cilia  and  loss   of   ciliary   motility   (S.   Suryavanshi   and   JG,  unpublished    data).    Among    these    enzymes,  Ttlll6Ap   has   been   well   characterized   biochemi-­
cally   and   found   to   have   an   exclusive   side   chain  elongase   activity   that   is   largely   restricted   to   E-­tubulin   (Janke   et   al.,   2005;;   van   Dijk   et   al.,   2007).  
GFP-­Ttll6Ap  localizes  mainly  to  cilia  (Janke  et  al.,  2005).   Thus,   selective   localization   of   side   chain  elongases   could   be   a   major   mechanism   that   is   re-­sponsible   for   differential   side   chain   length   distri-­bution   among   distinct   types   of   microtubules.      A  morpholino    knockdown    of    TTLL6    homolog   in  

12  原生動物学雑誌 第43巻 第1号 2010年  zebrafish   leads   to   shortening   of   olfactory   cilia  (Pathak   et   al.,   2007).   Thus,   tubulin   glutamylation  has   emerged   as   a   major   factor   that   contributes   to  the   assembly   and   function   of   cilia   and   basal   bod-­ies.    Recently,  enzymes  that  catalyze  glycylation,  G-­ligases,   were   identified   among   the   previously   un-­studied  members  of  the  TTLL  protein  superfamily.  
Rogowski    and    colleagues    showed    that    murine  TTLL3  and  TTLL10  act  as  G-­ligases  on  tubulin.  In  cultured  mammalian  cells,  the  murine  TTLL3  initi-­
ates  glycine  side  chains  that  are  subsequently  elon-­
gated   by   TTLL10   (Rogowski   et   al.,   2009).   Our  laboratory   has   identified   a   TTLL3   type   protein   of  Tetrahymena,   Ttll3Ap,   as   a   tubulin   G-­ligase   with  strong  initiating  activity  (Wloga  et  al.,  2009).  The  fruit  fly,  Drosophila  melanogaster  is  an  interesting  case,    because    this    species    lacks    an    obvious  TTLL10   elongase   homolog   and   TTLL3   type   pro-­teins  of  Drosophila  have  both  chain  initiation  and  elongation  G-­ligase  activity  on  tubulin  (Rogowski  et   al.,   2009).   In   Tetrahymena   and   Drosophila,  TTLL3   enzymes   are   required   for   tubulin   glycyla-­tion   in   vivo   (Rogowski   et   al.,   2009;;   Wloga   et   al.,  2009).    
Tetrahymena  has  6  genes  encoding  TTLL3  type  enzymes.   When   mildly   overexpressed   as   GFP   fu-­sions,  some  TTLL3  paralogs  colocalize  with  cilia,  some  with  basal  bodies  or  remain  in  the  cell  body  (Wloga   et   al.,   2009).   Surprisingly,   deletion   of   all  six  TTLL3  paralogs  resulted  in  viable  cells  that  are  motile   and   have   normal   morphology.    However  cilia   in   cells   lacking   TTLL3   activity   are   on   aver-­age   15%   shorter   as   compared   to   wildtype   cells.  
The    TTLL3    Tetrahymena    knockout    cells    have  residual   tubulin   glycylation   estimated   to   be   be-­tween   1-­8%   of   wildtype   (Wloga   et   al.,   2009).  
Since   all   TTLL3   G-­ligases   were   eliminated   in  these  strains,  another  tubulin  G-­ligase  with  an  ini-­
tiating   activity   must   exist.   This   enzyme   could   be-­long   to   less   conserved   classes   of   TTLLs   whose  members   are   present   in   Tetrahymena   (TTLL14,  TTLL15   and   TTLL16)   or   TTLL10   enzyme   that  acts   as   an   elongase   for   tubulin,   could   also   have  some   initiation   activity.   In   addition   to   a   mild   re-­duction  in  the  length  of  cilia,  TTLL3  Tetrahymena  knockout  cells  elevated  levels  of  acetyl-­K40  on  D-­tubulin   of   axonemes   (Wloga   et   al.,   2009).   It   thus  appears   that   TTLL3-­mediated   tubulin   glycylation  makes    axonemal    microtubules    more    dynamic,  which   in   turn   could   be   a   requirement   for   elonga-­tion  of  axonemes  to  a  proper  length.  This  result  fits  well  with  our  observation  that  katanin,  a  factor  that  promotes    microtubule    turnover,    is    required    for  ciliogenesis   (Sharma   et   al.,   2007).   While   the   im-­pact  of  TTLL3  on  the  axoneme  length  was  mild  in  Tetrahymena,  this  enzyme  appears  to  be  important  for   axoneme   assembly   in   multicellular   organisms.  
In   zebrafish,   a   morpholino   knockdown   of   TTLL3  led   to   dramatic   shortening   of   multiple   classes   of  cilia    (Wloga    et    al.,    2009)    and    RNAi-­mediated  knockdown    of    TTLL3    in    Drosophila    inhibited  assembly   of   sperm   axonemes   (Rogowski   et   al.,  2009).    
Another  clue  to  the  function  of  tubulin  glycyla-­tion   is   that   Tetrahymena   strains   lacking   TTLL3  genes  have  greatly  increased  levels  of  tubulin  glu-­
tamylation  on   axonemal   and   cortical   microtubules  (Wloga  et  al.,  2009).  Additional  experiments  have  confirmed   that   the   two   polymodifications   nega-­
tively  regulate  each  other.  Thus,  overexpression  of  E-­ligases  increased  the  levels  of  tubulin  glutamyla-­tion  (as  expected)  but  decreased  the  levels  of  tubu-­
lin  glycylation  on  the  same  microtubules  (Wloga  et  al.,  2009).  In  a  reverse  experiment,  overexpressing  a   TTLL3   G-­ligase   increased   the   levels   of   tubulin  glycylation   and   decreased   the   levels   of   tubulin  glutamylation  (Wloga  et  al.,  2009).  It  thus  appears  that  the  two  modifications  compete  with  each  other  in  vivo.  The  competition  could  be  a  result  of  utili-­
zation  of  the  same  Es  in  the  CTT  domain,  or  steric  inhibition    of    adjacent    polymodification    sites.  
While   tubulin   glutamylation   is   highly   conserved  and   present   in   all   eukaryotes   that   also   have   cilia  and  centrioles,  a  few  lineages  (e.g.  nematodes  and  trypanosomes)  lack  tubulin  glycylation.  It  is  possi-­

Jpn.  J.  Protozool.  Vol.  43,  No.  1.    (2010)

ble  therefore  that  the  main  role  of  tubulin  glycyla-­tion  is  to  negatively  regulate  tubulin  glutamylation  and   that   organisms   that   lack   tubulin   glycylation  could    have    evolved    additional    mechanisms    for  negative  regulation  of  tubulin  glutamylation.  
    SUMMARY    Recent  studies  brought  major  advances  in  eluci-­
dation   of   the   mechanisms   that   diversify   micro-­
tubules  at  the  subcellular  level.  Tubulin  polymodi-­
fications   have   emerged   as   a   major   determinant   of  assembly   and   functions   of   specific   microtubules,  and   in   particular,   those   forming   cilia   and   basal  bodies.  Studies  in  ciliates  have  contributed  to  iden-­tification   of   enzymes   that   polymodify   tubulin   and  uncovered   complex   regulatory   interactions   among  diverse  tubulin  modifications.    
    ACKNOWLEDGEMENT    I   am   grateful   to   the   Japanese   Society   of   Proto-­
zoology  and  Professor  Osamu  Numata  (University  of   Tsukuba)   for   inviting   me   to   present   a   lecture  during   the   JSP   annual   meeting   in   Tsukuba.   Our  recent   work   was   supported   by   a   National   Science  Foundation  grant  MBC-­033965.  
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Adoutte,   A.,   P.   Delgado,   A.   Fleury,   N.   Levilliers,  M.-­C.    Lainé,    M.-­C.    Marty,    E.    Boisvieux-­
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