1  

SUPPLEMENTARY  INFORMATION  

 

Supplementary  Table  1:  Cellular  factors  that  prevent  proteasome  binding  to  

K63-­‐conjugates  are  not  eluted  from  the  polyubiquitin  chains  by  high  salt  or  

detergent.  

 

Lysate wash buffer

Bound proteaosmes to UbnN4 after exposure to lysate or control wash buffer (%control ± SD)

No lysate Lysate

Control 100 ± 15 3 ± 0.6

900mM NaCl 146 ± 7 5 ± 0.4

1% Triton-X 100 137 ± 23 3 ± 0.3

0.1% SDS 99 ± 6 3 ± 0.4

 

 

The  GST-­‐ubiquitinated  Nedd4  was  incubated  with  or  without  the  HEK293  S100  

lysate  for  30  minutes,  and  then  all  samples  washed  with  TBSG  containing  either  

900mM  NaCl,  1%  Triton-­‐X  100,  or  0.1%  SDS.  The  resins  were  incubated  with  10nM  

26S  particles,  and  the  bound  proteasomes  measured  by  LLVY-­‐AMC  cleavage.  At  

concentrations  higher  than  1%  Triton-­‐X  100  or  0.1%  SDS  the  polyubiquitin  

conjugates  were  partially  eluted  from  the  resins,  preventing  further  analysis  of  

proteasome  binding  to  these  chains.  Proteasome  binding  is  expressed  as  percentage  

control  of  the  ubiquitinated  Nedd4  without  S100  lysate.  

   

  2  

Supplementary  Figure  1:  Schematic  diagram  of  experimental  designs.  

 

 

A-­‐C)  Schematic  representation  of  methods  used  in  the  experiments.  (A)  

Measurement  of  ubiquitin  conjugate  binding  to  proteasomes  within  crude  lysates  

(see  Figures  1A  and  S2C-­‐D).  (B)  Determining  the  effect  of  proteins  in  lysates  on  the  

binding  of  ubiquitin  conjugates  to  purified  proteasomes  (see  Figures  1B-­‐E,  S2E-­‐G).  

(C)  Identification  of  K48-­‐  and  K63-­‐specific  binding  proteins  (see  Figure  2).  

 

 

 

 

 

 

 

 

 

  3  

Supplementary  Figure  2:  Soluble  extracts  of  different  mammalian  cells  block  

K63-­‐conjugates  binding  to  the  proteasome,  and  this  inhibition  is  not  due  to  

deubiquitination.  

 

  4  

A)  Autoubiquitination  reactions  of  Nedd4  and  E6AP  form  ubiquitin  conjugates  of  

similar  lengths.  Representative  Coomassie  stained  gels  of  the  ubiquitinated  proteins  

(5%  of  total  sample  loaded).    

B)  Coomassie  stained  gel  of  forced  conjugation  of  K48Ub4  or  K63Ub4  to  Nedd4.  K48-­‐  

or  K63-­‐tetramers  were  forced  onto  GST-­‐Nedd4  by  incubating  the  resin  bound  E3  

ligase  with  E1,  E2  (UbcH5),  1mM  ATP  and  5µM  K48  or  K63Ub4.  10%  of  total  sample  

loaded.    

C,  D)  Nedd4-­‐K48n  conjugates  bind  proteasomes  in  a  rat  muscle  extract  preferentially  

to  Nedd4-­‐K63n  conjugates.  120µg  cell  extract  was  incubated  with  the  resin-­‐bound  

Nedd4  ubiquitin  conjugates,  and  the  bound  proteasomes  measured  by  their  LLVY-­‐

amc  peptidase  activity  (C).  (D)  Representative  immunoblot  of  the  resin-­‐bound  

conjugate  samples  used  in  for  ubiquitin  (C).  All  values  are  the  means  ±  SEM.  

E)  Cell  extracts  from  different  cells  can  prevent  proteasomes  binding  to  K63-­‐

ubiquitin  chains  similarly.  HEK293  cells  were  lysed  in  HEPES  buffer  with  or  without  

detergent  (0.5%  Triton-­‐X  100).  Rat  muscle  was  homogenised  in  TBSG.  Proteasome  

binding  was  measured  as  described  in  Figure  1.  

F,  G)  Endogenous  proteasomes  are  efficiently  depleted  from  the  extracts  by  

centrifugation.  Representative  analysis  by  immunoblot  of  20S  α-­‐subunits  (F)  and  

peptidase  activity  (G)  of  muscle  extracts  following  the  6-­‐hour  100,00xg  

centrifugation  to  deplete  the  endogenous  proteasome.  Sup=supernatant  and  

Pel=Pellet  after  the  6hr  centrifugation.  

H,  I)  K63-­‐polyubiquitin  conjugates  are  not  preferentially  deubiquitinated  to  K48-­‐

chains  in  the  cell  extract.  H)  Ubiquitin  immunblot  of  control  or  ubiquitinated  

  5  

substrates,  following  incubation  with  rat  muscle  lysate  and  26S  proteasomes  at  4°C  

for  30  minutes.  I)  Immunoblot  for  ubiquitinated  E6AP  or  Nedd4  with  or  without  

4mM  NEM  and  1mM  OPT  from  samples  used  in  Figure  1C.  

J)  Depletion  of  K48-­‐binding  proteins  from  the  cell  extract  does  not  affect  its  ability  of  

to  prevent  K63-­‐chains  binding  to  the  proteasome.  The  muscle  extract  was  first  

depleted  of  K48-­‐binding  proteins  by  incubation  with  polyubiquitinated  E6AP.  The  

unbound  fraction  of  the  lysate  (UbnE6  FT)  was  then  incubated  with  

polyubiquitinated  Nedd4  (UbnN4)  and  the  bound  proteasomes  measured.  

 

  6  

Supplementary  Figure  3:  Soluble  extracts  prevent  K63-­‐ubiquitinated  His-­‐Sic1  

from  binding  to  proteasomes.    

 

A)  K63-­‐polyubiquitinated  Sic1  is  rapidly  degraded  by  pure  proteasomes  in  vitro.  

Ubiquitinated  His-­‐Sic1  (40nM)  was  incubated  with  either  pure  26S  proteasomes  

(2nM),  the  proteasome-­‐depleted  cell  extract  (300µg  total  protein)  or  both  together  

for  15min  at  4°C  (0  min  time  point).  The  samples  were  then  incubated  at  37°C  for  

  7  

upto  40  min  with  (right)  or  without  (left)  NEM  and  οPT.  Polyubiquitinated  Sic1  was  

detected  by  immunoblot  (anti-­‐His  antibody).  26S  =  proteasome,  Lys  =  lysate.  

B)  K63-­‐Sic1  ubiquitin  conjugates  are  prevented  from  binding  to  proteasomes  by  a  

cell  extract.  Resin  bound  (NiNTA)  K63-­‐ubiquitinated  Sic1  (30nM)  was  incubated  

with  the  cell  extract  and  pure  proteasomes  (10nM)  for  30  min  at  4°C.  All  values  are  

the  means  ±  SEM.  

  8  

Supplementary  Figure  4:  Characterization  of  K48-­‐  and  K63-­‐specific  binding  

proteins.  

  9  

A)  USP5  binds  to  polyubiquitinated  E6AP  and  is  eluted  from  the  conjugates  by  the  

His-­‐UIM.  The  polyUb  E6AP  resin  was  incubated  with  the  HEK293  lysate  and  the  Ub-­‐

bound  proteins  eluted  with  the  His-­‐UIM  as  described.  The  UIM  elute  (left)  and  GST-­‐

resin  post  elution  (right)  were  separated  by  SDS-­‐PAGE  and  immunoblotted  for  

USP5.    

B)  Upon  gel  filtration  of  the  cell  extract,  the  UBD-­‐containing  proteins  identified  by  

MS  are  found  in  lysate  fractions  of  50-­‐400kDa  size.  The  rat  muscle  lysate  was  

separated  according  to  size  by  gel  filtration  as  in  Figure  1E.  The  fractions  were  then  

immunblotted  for  different  UBD-­‐containing  proteins  as  indicated.  

C)  Purity  of  the  bacterially  expressed  UBD-­‐containing  proteins.  Representative  

coomassie  stained  gels  of  purfied  STAM1,  Hrs,  TOM1,  hHR23A  and  hHR23B.  

 

 

  10  

Supplementary  Figure  5:  Hrs  prevents  polyubiquitinated-­‐Sic1  binding  to  the  

proteasome  but  the  STAM1-­‐VHS  domain  prevents  both  K48  and  K63-­‐

conjugates  binding  to  the  26S  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A)  Hrs  prevents  K63-­‐polyubiquitinated  Sic1  from  binding  to  the  proteasome.  Resin  

bound  K63-­‐Sic1  conjugates  (30nM),  Hrs  (50nM)  and  26S  proteasomes  were  

incubated  at  4°C  and  the  proteasomes  bound  to  the  conjugates  measured.  All  values  

are  the  means  ±  SEM.  

  11  

B)  The  STAM1-­‐VHS  domain  was  incubated  with  ubiquinated    E6AP  (solid  black  

lines)  or  Nedd4  (dashed  black  lines)  and  the  isolated  26S  proteasomes,  and  the  

proteasomes  bound  measured  in  the  usual  manner.  For  comparison,  the  effect  of  full  

length  STAM1  on  the  binding  of  K63-­‐conjugates  to  the  proteasome  is  also  shown  

(grey  lines).  The  purity  of  the  VHS-­‐STAM1  is  also  shown  by  coomassie  staining  

(right  panel).    

  12  

Supplementary  Figure  6:  hHR23B  does  not  stimulate  the  peptidase  activity  of  

the  26S  proteasome.  

 

 

 

 

 

 

 

 

 

 

hHR23B  does  not  affect  the  peptidase  activity  of  the  26S  proteasome.  The  ability  of  

polyubiqitin  conjugates  to  stimulate  proteasomal  peptidase  activity  (gate  opening)  

was  measured  as  previously  described  (Peth  et  al,  2009).  E6AP,  polyUb-­‐E6AP  or  

polyUb-­‐E6AP  and  hHR23B  (300nM)  were  incubated  with  pure  26S  proteasomes  

(2nM)  and  peptidase  activity  was  measured  with  GGL-­‐AMC  cleavage.  All  values  are  

the  means  ±  SEM.  

0

50

100

150

200

250

300

350

400

E6 UbnE6 UbnE6 hHR23B

GG

L-A

MC

cle

avag

e (%

con

trol

)

E6 UbnE6 UbnE6 hHR23B

  13  

Supplementary  Figure  7:  Determining  the  approximate  amounts  of  UBD-­‐

containing  proteins  in  the  muscle  lysate.  

 

       

 

 

 

 

 

 

 

Protein Amount

(ng protein/µµg lysate ±

SEM)

Concentration in lysate (nM ± SEM)

Crude estimate of Intracellular

Concentration (nM)

STAM1 0.58 ± 0.08 27.8 ± 3.9 1400

Hrs 0.07 ± 0.1 3.1 ± 1.4 200

hHR23B 0.21 ± 0.03 11.3 ± 0.8 600

hHR23A 0.09 ± 0.06 5.3 ± 1.8 300

Rel. FU/min µµg

protein

(LLVY-AMC peptidase activity)

Estimated amount of

26S

(ng protein/µµg lysate ±

SD)

Concentration in

lysate (nM ± SD)

Crude estimate of

Intracellular Concentration (nM)

S100 input 0.36 ± 0.04 2.7 ± 0.29 4 ± 0.5 200

Pure 26S 132 ± 7.2

B

0

2

4

6

8

10

0 2 4 6 8

Inte

nsity

(AU

)

pure protein (ng)

STAM1

0

2

4

6

8

0 1 2 3 4 In

tens

ity (A

U)

pure protein (ng)

0

20

40

60

80

0 2 4 6 8

Inte

nsity

(AU

)

pure protein (ng)

0 2 4 6 8

10 12 14

0 1 2 3 4

Inte

nsity

(AU

)

pure protein (ng)

Hrs

hHR23B hHR23A

C

D

  14  

 

A-­‐C)  Protein  content  of  hHR23B,  hHR23A,  STAM1  and  Hrs  in  the  rat  muscle  cell  

extract.  Indicated  amounts  of  the  pure  UBD-­‐containing  proteins  and  cell  extracts  

were  separated  by  SDS-­‐PAGE  and  immunoblotted  for  hHR23B,  hHR23A,  STAM1  and  

Hrs  (A).  The  intensities  of  the  bands  for  the  pure  proteins  were  quantified  using  

ImageJ  software,  and  plotted  against  the  known  pure  protein  amounts  to  generate  

standard  curves  for  each  of  the  UBD-­‐containing  proteins  (B).  The  intensities  of  the  

bands  specific  to  the  UBD-­‐containing  proteins  in  the  total  protein  lysates  were  then  

used  to  approximate  the  amount  of  the  Rad23  and  ESCRT0  proteins  in  the  lysate  (ng  

UBD-­‐containing  protein/µg  lysate)  (C).  The  concentration  of  the  UBD-­‐containing  

proteins  was  estimated  from  the  known  concentration  of  the  muscle  lysate  

(4mg/ml).  The  cellular  proteasome  content  was  measured  by  comparing  the  

proteasome  specific  peptidase  activity  of  the  lysate  with  the  activity  in  the  isolated  

26S  proteasomes  (D).  Crude  estimates  of  intracellular  protein  concentration  were  

calculated  from  the  assumption  that  the  total  protein  concentration  in  mammalian  

cells  is  between  200-­‐300mg/ml  (Ellis,  2001).  All  values  are  the  means  ±  SEM.  

 

  15  

Supplementary  Figure  8:  MHC  Class  I  molecules  depleted  of  β2m  bind  to  the  

proteasome,  but  a  reduction  in  cellular  ESCRT0  does  not  cause  proteasomal  

degradation  of  cell  surface  MHC  Class  I.  

 

  16  

A)  MHC  Class  I  molecules  are  not  degraded  following  siRNA-­‐mediated  depletion  of  

ESCRT  in  Hela-­‐K3  cells.  At  72  hours  after  siRNA  transfection  the  cells  were  treated  

with  20µM  MG132  for  three  hours.  Cell  surface  MHC  Class  I  molecules  were  

immunoprecipitated  as  previously  described.  Immunoblots  of  immunoprecipitated  

cell  surface  Class  I  (top  panel)  and  lysates  for  STAM1  and  Hrs  (middle  panels)  are  

shown.  β-­‐actin  (lower  panel)  served  as  a  loading  control.  

B)  Misfolded  MHC  Class  I  molecules  associate  with  the  proteasome  following  β2m  

siRNA-­‐mediated  depletion.  HeLa  and  HeLa-­‐K3  cells  were  transfected  with  siRNA  

targeting  STAM1  and  Hrs  or  β2m.  After  72  hours  the  cells  were  treated  with  or  

without  20µM  MG132  for  three  hours.  The  cells  were  then  lysed  and  the  26S  

proteasomes  isolated  using  the  Ubl-­‐affinity  method.  Class  I  molecules  bound  to  the  

proteasomes  were  analysed  by  immunoblot  (top  panel).  The  immunoblot  of  the  20S  

α-­‐subunits  confirms  the  affinity  isolation  of  the  26S  proteasomes  (bottom  panel).  H-­‐

K3=HeLa-­‐K3.  

C)  ESCRT0  is  required  for  the  lysosomal  degradation  of  MHC  Class  I  molecules  in  

Hela-­‐K3  cells.    ESCRT0  was  reduced  in  HeLa-­‐K3  cells  by  siRNA  to  Hrs  and  STAM.  72  

hrs  later,  the  cells  were  fixed  and  the  levels  of  STAM  and  MHC  Class  I  visualised  by  

confocal  microscopy.  

D)  After  an  ESCRT0  knockdown  some  26S  proteasomes  are  found  in  association  

with  recycling  endosomes  and  MHC  Class  I  molecules.  HeLa-­‐K3  cells  were  incubated  

with  the  W6/32  antibody  or  fluorescently  tagged  transferrin  (AlexoFluor  594-­‐

Transferrin)  for  15  min.  The  cells  were  fixed  with  4%  paraformaldehyde  and  26S  

proteasomes  visualised  with  an  anti-­‐β5  antibody.  

  17  

EXPERIMENTAL  PROCEDURES  

Plasmids  and  antibodies  

The  clone  for  TOM1  (IMAGE  Clone  6470390)  was  purchased  from  Open  Biosystems,  

and  cloned  into  the  bacterial  expression  vector  pET15b  using  the  following  primers:  

Tom1  NdeI  For  GGAATTCCATATGATGGACTTTCTCCTGGGGAACCCGTT  and  Tom1  

XhoI  Rev  CCGCTCGAGGACCCCACACTCATAAGGCAAACAGC.  The  GST-­‐Nedd4  and  

GST-­‐E6AP  plasmids  were  generously  provided  by  Allan  Weissman  (National  Cancer  

Institute).  The  E6AP  construct  lacks  the  first  32  residues  and  the  Nedd4  encodes  the  

full-­‐length  protein  without  the  calcium  binding  C2  domain.  GST-­‐Hrs  (pGEX4T2-­‐Hrs),  

and  His-­‐STAM1  (pTrcHisA-­‐Hbp/STAM1)  were  gifts  from  Sylvie  Urbé  (University  of  

Liverpool).  GST-­‐hHR23A  and  GST-­‐hHR23B  were  kind  gifts  from  Peter  Howley  

(Harvard  Medical  School).  The  following  antibodies  were  used:  mouse  monoclonal  

to  Hrs  (Enzo  Life  Sciences),  rabbit  polyclonal  to  STAM  (Santa  Cruz),  rabbit  

polyclonal  to  hHR23A  (Bethyl  Laboratories),  rabbit  polyclonal  to  hHR23B  (Bethyl  

Laboratories  and  Enzo  Life  Sciences),  rabbit  polyclonal  to  the  20S  Beta5  subunit  

(PW8895,  Enzo  Life  Sciences)  and  mouse  monoclonals  to  ubiquitin  (FK1,  Enzo  Life  

Sciences  and  P4D1,  Santa  Cruz).  All  MHC  Class  I  antibodies  were  kind  gifts  of  Paul  

Lehner  (Cambridge  Institute  for  Medical  Research).  Rabbit  antiserum  to  TOM1  was  

kindly  given  by  Wanjin  Hong  (Institute  of  Molecular  and  Cell  Biology,  Singapore).  

The  AlexoFluor594-­‐transferrin  was  given  by  Margaret  Robinson  (Cambridge  

Institute  for  Medical  Research).  

 

 

  18  

Purification  of  UBD-­‐containing  proteins  

  GST  conjugated  Hrs,  hHR23A  and  hHR23B  were  expressed  in  BL21DE3star  E.  

Coli,  and  purified  using  glutathione  columns  and  FPLC  (Biorad).  The  GST  tag  was  

then  cleaved,  using  thrombin  or  Prescission  protease  (GE  Healthcare),  and  removed  

by  incubation  with  GSH  sepharose  (GE  Healthcare).  The  eluted  proteins  were  

further  purified  by  ion-­‐exchange  chromatography,  dialysed,  and  stored  in  25mM  

HEPES,  40mM  KCL,  1mM  DTT  and  10%  glycerol.  His-­‐tagged  proteins  were  similarly  

expressed  in  E.  Coli  and  purified  using  a  NiNTA  column  and  FPLC,  but  the  His  tag  

was  not  removed.  

 

Mass  spectrometry  analysis      

  For  samples  separated  by  SDS-­‐PAGE,  gel  bands  of  interest  were  excised,  cut  

into  ~1mm3  pieces  and  destained  in  a  50%  (v/v)  methanol,  50  mM  NH4HCO3  buffer  

for  Coomassie  stained  gels.  Silver  stained  gel  pieces  were  destained  using  the  

BioRad  silver  stain  destaining  kit.  Destained  gel  pieces  were  dehydrated  with  

acetonitrile  for  15  minutes  and  dried  in  vacuuo  for  5  minutes.  Proteins  were  

digested  in-­‐gel  by,  firstly,  allowing  dehydrated  gel  pieces  to  absorb  an  ice-­‐cold  5  

ng/uL  trypsin  in  50  mM  NH4HCO3  solution  on  ice,  then  incubated  at  37  °C  overnight.  

Digested  peptides  were  extracted  by  a  50%  (v/v)  acetonitrile,  5%  (v/v)  formic  acid  

buffer,  followed  by  100%  acetonitrile.  Samples  were  acidified  to  pH  2-­‐3  with  formic  

acid,  desalted  by  C18  solid-­‐phase  extraction  (3M  Empore),  dried  in  vacuuo,  

resuspended  in  a  5%  (v/v)  acentonitrile,  4%  (v/v)  formic  acid  buffer,  and  analysed  

by  LC-­‐MS/MS  on  a  LTQ-­‐Orbitrap  XL  (Thermo  Fischer  Scientific)  equipped  with  a  

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Famos  autosampler  (LC  Packings)  and  an  Agilent  1100  binary  HPLC  pump  (Agilent  

Technologies).  Peptides  were  separated  on  a  100  µm  I.D.  microcapillary  column  

packed  first  with  20  cm  of  Magic  C18  resin  (5  µm,  100  Å,  Michrom  Bioresources).  

Separation  was  achieved  through  applying  a  gradient  from  5  to  35%  acetonitrile  in  

0.125%  formic  acid  over  33  min  at  approximately  300nl/min.  Electrospray  

ionization  was  enabled  through  applying  a  voltage  of  1.8  kV  through  a  PEEK  

junction  at  the  inlet  of  the  microcapillary  column.    

  For  whole  samples  that  were  TCA  precipitated,  protein  pellets  were  

resuspended  in  a  100mM  NH4HCO3,  10%  (v/v)  acetonitrile  buffer  and  digested  

using  a  1:50  ratio  of  trypsin:protein  at  37  °C  overnight.  The  digest  was  quenched  

with  a  50%  (v/v)  acetonitrile,  5%  (v/v)  formic  acid  buffer,  desalted  analysed  by  LC-­‐

MS/MS  as  described  above.  

  All  proteins  identified,  including  single  peptide  hits  that  were  manually  

verified,  are  detailed  in  the  Supplementary  file.  

 

Immunofluorescence  

HeLa-­‐K3  cells  were  fixed  with  4%  paraformaldehyde  for  15  min  and  then  

permeabilized  for  5  min  in  0.2%  Triton-­‐X  100  PBS.  Cells  were  blocked  for  15  min  in  

0.2%  BSA  PBS  before  appropriate  staining  with  primary  and  secondary  antibodies.  

Images  were  visualised  using  a  Zeiss  LSM710  Confocal  Microscope.  The  transferrin  

uptake  experiments  were  performed  as  described,  except  that  the  cells  were  

deprived  of  serum  for  30  min  prior  to  a  15  min  incubation  with  the  AlexoFluor594-­‐

transferrin  and  the  W6/32  Class  I  antibody.