Rqc2p and 60S ribosomal subunits
mediate mRNA-independent
elongation of nascent chains
RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged
1
Department of Biochemistry, University of Utah, UT 84112,
tRNA to the A site
and directs the elongation of nascent chains independently of mRNA or
USA. 2Department of Biochemistry, Stanford University, Palo
Cellular and Molecularmechanism of protein synthesis, in which
Alto,work
CA 94305,
USA. 3Department
40S subunits. Our
uncovers
anofunexpected
Pharmacology, University of California, San Francisco, San
4
a protein—not an
mRNA—determines
tRNA
recruitment
and the tagging of nascent
Francisco, CA 94158, USA. Howard Hughes
Medical
Institute,
University of California, San
Francisco,
San
Francisco,
CA
chains with carboxy-terminal
Ala
and
Thr
extensions
(“CAT
tails”).
5
D
94158, USA. California Institute for Quantitative Biomedical
D
figs.
S1 to S7).
Tif6p
waswhich
not observed
complex
(RQC),
mediates thebound to
ity control
ubiquitylation
degradation
of incompletely
the
same 60Sand
particles
bound
by RQC factors
synthesized
nascent
(1–4). the
The molecular
(figs.
S1 to S3).
Wechains
repeated
purification, imcomponents of the RQC include the AAA adenaging,
and 3D classification from rqc2D cells and
osine triphosphatase Cdc48p and its ubiquitincomputed
difference
maps. This analysis
binding cofactors,
the RING-domain
E3 ligase did not
Ltn1p,density
and twoattributable
proteins of unknown
function,
reveal
to Rqc1p
but did idenRqc1p
and
Rqc2p.
We
set
out
to
determine
the
tify Rqc2p as a transfer RNA (tRNA)–binding
mechanism(s) by which relatively rare (5) proprotein
that
occupies
40S binding
teins such
as Ltn1p,
Rqc1p, the
and Rqc2p
recognize surface
and
the 60S
elongated
that meets
andLtn1p
rescue as
stalled
ribosomemolecule
nascent chain
complexes,
which
are vastly outnumbered
riRqc2p
at the
sarcin-ricin
loop (SRL)by(Figs.
1 and
bosomes translating normally or in stages of
2 and
figs.
S1
to
S5).
Comparison
of
the
60S-bound
assembly.
Ltn1p
of isolated
Ltn1p sugTo with
reducereconstructions
structural heterogeneity
and enrich
for complexes
occupied byof
stalled
nascent
gests
that thestill
N terminus
Ltn1p
engages the
chains,
we immunoprecipitated
RQCregion—
SRL
with
Rqc2p and thatRqc1p-bound
the middle
assemblies from Saccharomyces cerevisiae strains
which contains long HEAT/Armadillo repeats
1
that
adoptofan
elongated
superhelical
structure—
Department
Biochemistry,
University
of Utah, UT 84112,
USA. 2Department
of Biochemistry,
Stanford
University,
Palo
reaches
around
the
60S
(6).
This
conformation
Alto, CA 94305, USA. 3Department of Cellular and Molecular
probably
positions
C-terminal
RING
domain
Pharmacology,
University ofthe
California,
San Francisco,
San
Francisco, CA 94158, USA. 4Howard Hughes Medical Institute,
near
the exit tunnel to ubiquitylate stalled nasUniversity of California, San Francisco, San Francisco, CA
94158,
USA. 5California
for Quantitative
cent
chains
[figs.Institute
S5 and
S6 and Biomedical
(7)].
Research, University of California, San Francisco, San
A refined
reconstruction
of Systems
the Rqc2p-occupied
Francisco,
CA 94158,
USA. 6Center for RNA
Biology,
University
of California, San Francisco,
San Francisco,
CA extensive
class
demonstrated
that
Rqc2p
makes
94158, USA. 7Department of Biochemistry and Biophysics,
contacts
with
an San
approximately
P-site
University of
California,
Francisco, San Francisco,
CA positioned
94158, USA. 8Institute for Cellular and Molecular Biology,
(~P-site)
tRNA
(Figs.
1
and
2
and
fig.
S7). Rqc2p
University of Texas at Austin, Austin, TX 78712, USA.
9
Department
of
Molecular
Biosciences,
University
of
Texas
at contact
has a long coiled coil10 that makes direct
Research, University of California, San Francisco, San
lacking the C-terminal RING domain of Ltn1p,
espite the processivity
of protein syntheFrancisco, CA 94158, USA. 6Center for RNA Systems Biology,
University of or
California,
San Francisco,
San Francisco, CA
which prevents substrate ubiquitylation and
sis, faulty messages
defective
ribosomes
94158, USA. 7Department of Biochemistry and Biophysics,
Cdc48 recruitment (1). Three-dimensional (3D)
can result inUniversity
translational
stalling
and
inof California, San Francisco, San Francisco, CA
8
Institute
for
Cellular
and
Molecular
Biology,
94158,
USA.
classification of Ltn1DRING particles revealed
complete nascent chains. In Eukarya, this
University of Texas at Austin, Austin, TX 78712, USA.
Austin, Austin, TX 78712, USA. Mass Spectrometry and
9
leads to recruitment
the ribosome
qual- of Texas at
Department ofof
Molecular
Biosciences, University
Proteomics Core Facility, University of Utah, UT 84112, USA.
10
Austin,
Austin, TX
78712, USA.
Mass Spectrometry
Fig. 1. Cryo-EM reconstructions of peptidyl-tRNA-60S ribosomes
boundSchool
by the
RQC
components
*Present address:
of Life
Sciences,
Tsinghua University,
(RQC),
which
mediates
the and
ity control complex
Proteomics Core Facility, University of Utah, UT 84112, USA.
Beijing
China. †Corresponding
Rqc2p and Ltn1p. (A) A peptidyl-tRNA-60S complex isolated
by 100084,
immunoprecipitation
of author.
Rqc1p.E-mail:
The jonathan.
ubiquitylation and*Present
degradation
incompletely
address: Schoolof
of Life
Sciences, Tsinghua University,
[email protected] (J.S.W.), [email protected] (O.B.), adam.
ribosome density is transparent to visualize the nascent chain. (B) Rqc2p (purple) and an ~A-site tRNA
[email protected] (A.F.)
Beijing 100084, China. †Corresponding author. E-mail: jonathan.
synthesized nascent
chains (1–4). The molecular
(yellow) bound to peptidyl-tRNA-60S complexes. Landmarks are indicated (L1, L1 stalk; SB, P-stalk base).
[email protected] (J.S.W.), [email protected] (O.B.), adam.
components of [email protected]
RQC include
the AAA aden(C) Ltn1p (tan) bound to Rqc2p-peptidyl-tRNA-60S complexes (B).
(A.F.)
SCIENCE sciencemag.org
osine triphosphatase Cdc48p and its ubiquitinbinding cofactors,SCIENCE
the RING-domain
sciencemag.org E3 ligase
2 JANUARY 2015 • VOL 347 ISSUE 6217
75
Ltn1p, and two proteins of unknown function,
Rqc1p and Rqc2p. We set out to determine the
mechanism(s) by which relatively rare (5) proteins such as Ltn1p, Rqc1p, and Rqc2p recognize
and rescue stalled 60S ribosome nascent chain
loaded from www.sciencemag.org on January 1, 2015
D
lacking the C-terminal RING domain of Ltn1p,
espite the processivity of protein synthewhich prevents substrate ubiquitylation and
sis, faulty messages or defective ribosomes
Cdc48 recruitment (1). Three-dimensional (3D)
can result in translational stalling and inclassification of Ltn1DRING particles revealed
complete nascent chains. In Eukarya, this
leads to recruitment of the ribosome quality control complex (RQC), which mediates the
ubiquitylation and degradation of incompletely
synthesized nascent chains (1–4). The molecular
components of the RQC include the AAA adenosine triphosphatase Cdc48p and its ubiquitinE3 ligase
1 binding cofactors,2 the RING-domain
Peter S. Shen, Joseph
Yidan
Qin,8,9 function,
Xueming Li,7* Krishna Parsawar,10
Ltn1p, andPark,
two proteins
of unknown
3,4,5,6
1,10
7
Matthew H. Larson,
James
Cox,
Yifan Cheng,
Alan M. Lambowitz,8,9
Rqc1p and Rqc2p.
We set
out to determine
the
3,4,5,6
2
mechanism(s) by which
relatively
rare (5) proJonathan S. Weissman,
† Onn
Brandman,
† Adam Frost1,7†
teins such as Ltn1p, Rqc1p, and Rqc2p recognize
and rescue stalled 60S ribosome nascent chain
In Eukarya, stalled
translation
40S dissociation
and recruitment of the ribosome
complexes,
which areinduces
vastly outnumbered
by ribosomes translating
normally
or insubunit,
stages of which mediates nascent chain
quality control complex
(RQC) to
the 60S
assembly.
degradation. Here
we report cryo–electron microscopy structures revealing that the
reduce structural heterogeneity and enrich
RQC components To
Rqc2p
(YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the
for complexes still occupied by stalled nascent
60S subunit at sites
exposed
after 40S
dissociation,
placing the Ltn1p RING (Really
chains, we immunoprecipitated
Rqc1p-bound
RQC
Interesting New assemblies
Gene) domain
near the
exit channel
and Rqc2p over the P-site transfer
from Saccharomyces
cerevisiae
strains
Ltn1p with
reconstructions
isolated
Ltn1p
sugdegradation.
Hereofwe
report
cryo–electron
microscopy structur
gests that
thecomponents
N terminus ofRqc2p
Ltn1p (YPL009C/Tae2)
engages the
RQC
and Ltn1p (YMR247C
SRL with
Rqc2p
and at
that
the exposed
middle region—
60S
subunit
sites
after 40S dissociation, placing th
which contains
longNew
HEAT/Armadillo
Interesting
Gene) domainrepeats
near the exit channel and Rqc2p
RNA
Wesuperhelical
further
Rqc2p
alanin
that adopt
an(tRNA).
elongated
structure—
RE Sdemonstrate
EAR
CH |that
RE
P O R recruits
TS
to the
the60S
A site
the elongation of nascent chains
reachestRNA
around
(6).and
Thisdirects
conformation
40S
subunits.
Our work RING
uncovers
an unexpected mechanism of
probably
positions
the C-terminal
domain
protein—not
an mRNA—determines
near thea exit
tunnel to ubiquitylate
stalled nas- tRNA recruitment and the
60S
ribosomes
with
the exit (“CAT tails
chains
with
carboxy-terminal
Alachains
and Thrinextensions
cent chains
[figs.
S5 and
S6
and nascent
(7)].
A refined
of
the
Rqc2p-occupied
tunnelreconstruction
and
extraribosomal
densities
(Fig.
1).
lacking the C-term
espite the processivity of protein synthe- These
class extraribosomal
demonstrated
thatmessages
Rqc2p
makes
extensive
features
were
resolved
which prevents s
sis, faulty
or defective
ribosomesbetween
contacts with an
P-site positioned
canapproximately
result
in translational
stalling
and
in- or Cdc48
5
and
14Å
and
proved
to
be
either
Tif6p
RQC recruitmen
(~P-site) tRNAcomplete
(Figs. 1 and
2 andchains.
fig. S7).
classification of L
nascent
InRqc2p
Eukarya, this
components
as
characterized
below
(Fig.
1
and
has a long coiled
that makes direct
contact qualleadscoil
to recruitment
of the ribosome
Downloaded from www.sciencemag.org on January 1, 2015
PROTEIN
quality control complex (RQC) to the 60S subunit, which mediates nascent chain
degradation. Here we report cryo–electron microscopy structures revealing that the
RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the
60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really
Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer
RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged
tRNA to the A site and directs the elongation of nascent chains independently of mRNA or
40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which
a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent
SYNTHESIS
chains with carboxy-terminal Ala and Thr extensions (“CAT tails”).
Fig. 1. Cryo-EM re
Rqc2p and Ltn1p.
ribosome density is
(yellow) bound to p
(C) Ltn1p (tan) bou
mutations in the mammalian
N1 cause neurodegeneration in
rly, mice with mutations in a centem–specific isoform of tRNAArg
homolog of yeast Hbs1 which
OTA/Dom34 to dissociate stalled
suffer from neurodegeneration
rvations reveal the consequences
talls impose on the cellular ecoia rescue stalled ribosomes with
senger RNA (tmRNA)–SmpB sysnds nascent chains with a unique
hat
targets the incomplete proProc. Natl. Acad. Sci. U.S.A. 107,
proteolysis
(23). The mechanisms
).
Aravind,
RNA Biol.
360–372 to recotes,
which
lack11,tmRNA,
ue stalled ribosomes and their
Natl. Acad. Sci. U.S.A. 106, 2097–2103
nslation products have been un—and
Rqc2p’s
CAT
tail tagging
Science 345,
455–459
(2014).
Sauer,
Annu. Rev. Biochem.
101–124
articular—bear
both76,
similarities
o the tmRNA trans-translation
ean, T. Melcher, W. Keller, EMBO J. 17,
lutionary convergence upon dismsNucleic
for extending
Acids Res. 35,incomplete
6740–6749 nashe C terminus argues for their
n, J. M. Chandonia,
S. E. Brenner,One
Genome
maintaining
proteostasis.
ad(2004).
ng stalled chains is that it may
from
EN
TS normal translation products
ir performed
removalatfrom
the protein
as
the University
of Utahpool.
and
rnia.
We thank D.exclusive,
Belnap (University
of
ot
mutually
possibility
(University
of California,
San Francisco)
for
sion
serves
to test the
functional
ron microscopes; A. Orendt and the Utah
emance
ribosomal
subunits,
soExtreme
that the
Computing
and the NSF
nd
disposeEnvironment
of defective
large for
subg Discovery
consortium
Sidote (University of Texas at Austin)
eD.stalling.
4780–4789 (1998).
25. K. W. Gaston et al., Nucleic Acids Res. 35, 6740–6749
(2007).
26. G. E. Crooks, G. Hon, J. M. Chandonia, S. E. Brenner, Genome
Res. 14, 1188–1190 (2004).
The authors declare no competing financial int
cryo-EM structures have been deposited at the
Microscopy Data Bank (accession codes 2811,
6171, 6172, 6176, and 6201).
SUPPLEMENTARY MATERIALS
ACKN OWLED GMEN TS
Electron microscopy was performed at the University of Utah and
the University of California. We thank D. Belnap (University of
Utah) and M. Braunfeld (University of California, San Francisco) for
supervision of the electron microscopes; A. Orendt and the Utah
Center for High Performance Computing and the NSF Extreme
Science and Engineering Discovery Environment consortium for
computational support; D. Sidote (University of Texas at Austin)
www.sciencemag.org/content/347/6217/75/su
Materials and Methods
Figs. S1 to S13
Table S1
References (27–41)
7 August 2014; accepted 14 November 2014
10.1126/science.1259724
for help processing RNA-seq data; and D. Herschlag and
P. Harbury for helpful comments. Amino acid analysis was
performed by J. Shulze at the University of California, Davis
CANCER
Proteomics
Core.ETIOLOGY
Edman sequencing was performed at Stanford
University’s Protein and Nucleic Acid Facility by D. Winant. This
work was supported by the Searle Scholars Program (A.F.);
Stanford University (O.B.); NIH grants 1DP2GM110772-01 (A.F.),
GM37949, and GM37951 (A.M.L.); the Center for RNA Systems
Biology grants P50 GM102706 (J.S.W.) and U01 GM098254
(J.S.W.); and the Howard Hughes Medical Institute (J.S.W.).
The authors declare no competing financial interests. The
cryo-EM structures have been deposited at the Electron
Microscopy Data Bank (accession codes 2811, 2812, 6169, 6170,
6171, 6172, 6176, and 6201).
Variation in cancer risk among
tissues can be explained by the
number of stem cell divisions
1
SUPPLEMENTARY
MATERIALS
Cristian Tomasetti
* and
Bert Vogelstein2*
www.sciencemag.org/content/347/6217/75/suppl/DC1
Materials
Methods
Some and
tissue
types give rise to human cancers millions of
Figs. S1 to S13
tissue types. Although this has been recognized for more
Table S1
explained.
Here, we show that the lifetime risk of cancers
References
(27–41)
times more often than
than a century, it has
of many different type
correlated (0.81) with the total number of divisions of the normal self-renewing
7 August 2014; accepted 14 November 2014
maintaining that tissue’s homeostasis. These results suggest that only a third of
10.1126/science.1259724
in cancer risk among tissues is attributable to environmental factors or inherite
t of Biochemistry, University of Utah, UT 84112,
rtment of Biochemistry, Stanford University, Palo
305, USA. 3Department of Cellular and Molecular
gy, University of California, San Francisco, San
CA 94158, USA. 4Howard Hughes Medical Institute,
f California, San Francisco, San Francisco, CA
. 5California Institute for Quantitative Biomedical
niversity of California, San Francisco, San
CA 94158, USA. 6Center for RNA Systems Biology,
f California, San Francisco, San Francisco, CA
. 7Department of Biochemistry and Biophysics,
f California, San Francisco, San Francisco, CA
. 8Institute for Cellular and Molecular Biology,
f Texas at Austin, Austin, TX 78712, USA.
t of Molecular Biosciences, University of Texas at
tin, TX 78712, USA. 10Mass Spectrometry and
Core Facility, University of Utah, UT 84112, USA.
dress: School of Life Sciences, Tsinghua University,
84, China. †Corresponding author. E-mail: jonathan.
ucsf.edu (J.S.W.), [email protected] (O.B.), adam.
du (A.F.)
E sciencemag.org
lacking the C-terminal RING domain of Ltn1p,
which prevents substrate ubiquitylation and
Cdc48 recruitment (1). Three-dimensional (3D)
classification of Ltn1DRING particles revealed
class demonstrated that Rqc2p makes extensive
contacts with an approximately P-site positioned
(~P-site) tRNA (Figs. 1 and 2 and fig. S7). Rqc2p
has a long coiled coil that makes direct contact
Fig. 1. Cryo-EM reconstructions of peptidyl-tRNA-60S ribosomes bound by the RQC components
Rqc2p and Ltn1p. (A) A peptidyl-tRNA-60S complex isolated by immunoprecipitation of Rqc1p. The
ribosome density is transparent to visualize the nascent chain. (B) Rqc2p (purple) and an ~A-site tRNA
(yellow) bound to peptidyl-tRNA-60S complexes. Landmarks are indicated (L1, L1 stalk; SB, P-stalk base).
(C) Ltn1p (tan) bound to Rqc2p-peptidyl-tRNA-60S complexes (B).
2 JANUARY 2015 • VOL 347 ISSUE 6217
75
Downloaded from www.sciencemag.org
pite the processivity of protein synthe, faulty messages or defective ribosomes
n result in translational stalling and inmplete nascent chains. In Eukarya, this
ds to recruitment of the ribosome qualol complex (RQC), which mediates the
ation and degradation of incompletely
ed nascent chains (1–4). The molecular
nts of the RQC include the AAA adenphosphatase Cdc48p and its ubiquitincofactors, the RING-domain E3 ligase
nd two proteins of unknown function,
nd Rqc2p. We set out to determine the
sm(s) by which relatively rare (5) proh as Ltn1p, Rqc1p, and Rqc2p recognize
ue stalled 60S ribosome nascent chain
es, which are vastly outnumbered by ritranslating normally or in stages of
.
uce structural heterogeneity and enrich
lexes still occupied by stalled nascent
e immunoprecipitated Rqc1p-bound RQC
es from Saccharomyces cerevisiae strains
he large
and refactors
e tRNA
the andomain,
ns along
To test these predictions, we expressed a series
of reporters containing a stalling sequence [tracts
of up to 12 consecutive arginine codons, including pairs of the difficult-to-decode CGA codon
(17)], inserted between the coding regions of green
fluorescent protein (GFP) and red fluorescent
different phenotypes: Expression of the stalling
reporter in ltn1D led to the formation and accumulation of higher-molecular-weight species
that resolve as a smear ~1.5 to 5 kD above the
expected position of GFP (Fig. 4A). GFP massshifted products are observable in rqc1Dltn1D
specific
NA after
ct RQC2
y a new
tron reresence
ment of
QC (Fig.
ns with
are the
though
of other
s specio direct
ions 32
hich are
enosine
GC)
and
(11–13),
reverse
Further
ed that
minated
d reads
Fig. 2. Rqc2p binding to the 60S ribosome and ~P-site, and ~A-site tRNAs. (A) Rqc2p contacts ~Pand ~A-site tRNAs, the SRL, and P-stalk base ribosomal RNA (SB). (B) Rigid body fitting of tRNAs structures (ribbons) into EM densities (mesh).
and positions 32
some of which are
Fig. 3). Adenosine
tRNAAla(AGC) and
to inosine (11–13),
osine upon reverse
B and C). Further
data revealed that
s also deaminated
2p-enriched reads
with the structure,
nds to the D-, T-,
site tRNA, and that
36 edited motif acor these two tRNAs
dine at position 36
ation between the
loops that harbor
2p evolved to bind
es, we considered
structural and bioRqc2p binds the
bosome dissociates
ascent chains accueight species in the
s absence [Fig. 4A,
Finally, amino acid
an be mediated by
vitro even when delate and the small
acts led us to hypoote the extension of
alanine and threoon reaction that is
pothesis makes speqc2p-dependent inght of the nascent
C terminus excluISSUE 6217
Fig. 2. Rqc2p binding to the 60S ribosome and ~P-site, and ~A-site tRNAs. (A) Rqc2p contacts ~Pand ~A-site tRNAs, the SRL, and P-stalk base ribosomal RNA (SB). (B) Rigid body fitting of tRNAs structures (ribbons) into EM densities (mesh).
Fig. 3. Rqc2p-dependent enrichment of tRNAAla(IGC) and tRNAThr(IGU). (A) tRNA cDNA reads extracted from
purified RQC particles and summed per unique anticodon,with versus without Rqc2p. (B) Secondary structures of
tRNAAla(IGC) and tRNAThr(IGU). Identical nucleotides are underlined. Edited nucleotides are indicated with asterisks
(24, 25). (C) Weblogo representation of cDNA sequencing reads related to shared sequences found in anticodon
loops (positions 32 to 38) of mature tRNAAla(IGC) and tRNAThr(IGU) (26). (D) ~A-tRNA contacts with Rqc2p at
the D-,T-, and anticodon loops. Identical nucleotides between tRNAAla(IGC) and tRNAThr(IGU) are colored as in (B)
(A, green; U, red; C, blue; G, orange) and pyrimidine, purple. Anticodon nucleotides are indicated as slabs.
sciencemag.org SCIENCE
ribozyme. The ribozyme cleaves the coding
sequence of the GFP mRNA, leaving a truncated non-stop mRNA that causes a stall during
translation of its final codon [fig. S8 (19)]. Thus,
the GFP mass shift is located at or after the stall
sequence but cannot be explained by mRNA
translation past the stalling tract in any frame.
of LTN1) led to a robust heat shock response
that is fully dependent on Rqc2p, although the
mechanism by which Rqc2p enabled this stress
response was unclear. We hypothesized that
CAT tails may be required for activation of heat
shock factor 1 (Hsf1p). To isolate the effect of
CAT tails in this context, we sought an rqc2
activation.
Integrating our observations, we propose the
model schematized in fig. S13. Ribosome stalling
leads to dissociation of the 60S and 40S subunits, followed by recognition of the peptidyltRNA-60S species by Rqc2p and Ltn1p. Ltn1p
ubiquitylates the stalled nascent chain, and this
Fig. 4. Rqc2p-dependent formation of CAT
tails. (A, B, and D) Immunoblots of stalling
reporters in RQC deletion strains. (C) Total
amino acid analysis of immunoprecipitated GFP
expressed in ltn1D and ltn1Drqc2D strains,
n = 3 independent immunoprecipitations.
(E) Triplicate GFP levels measured with a flow
cytometer and normalized to a wild-type
control. EV, empty vector. All error bars are
standard deviations.
SCIENCE sciencemag.org
2 JANUARY 2015 • VOL 347 ISSUE 6217
77
Materials and Methods
S. cerevisiae strains
Strain
Background
yOBj065
BY4741
yOBj067
BY4741 ltn1::kan
yOBj068
BY4741 rqc1::nat
yOBj069
BY4741 rqc2::kan
yOBj070
yOBj072
yOBj071
BY4741 ltn1::kan
rqc2::nat
BY4741 rqc1::nat
rqc2::kan
BY4741 rqc1::nat
ltn1:kan
yOB520
BY4741 ltn1::kan
yOBj020
BY4741 ltn1::kan
rqc2::nat
yOBj073
BY4741 ltn1::kan
yOBj043
BY4741
yOBj047
BY4741 rqc2::kan
yOBj048
BY4741 rqc2::kan
yOBj049
BY4741 rqc2::kan
yOBj051
BY4741 ltn1::kan
rqc2::nat
yOBj052
BY4741 ltn1::kan
rqc2::nat
yOBj053
BY4741 ltn1::kan
rqc2::nat
yOB255
yOBj059
BY4741
ura3::HSEEmGFP
BY4741
ura3::HSEEmGFP
rqc2::kan
rqc1::nat
rps0a::hyg
Plasmid 1
pTDH3-GFP_R12_RFP,
URA3 marker
pTDH3-GFP_R12_RFP,
URA3 marker
pTDH3-GFP_R12_RFP,
URA3 marker
pTDH3-GFP_R12_RFP,
URA3 marker
pTDH3-GFP_R12_RFP,
URA3 marker
pTDH3-GFP_R12_RFP,
URA3 marker
pTDH3-GFP_R12_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
pTDH3GFP_R12_TEV_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
pTDH3GFP_TEV_R12_RFP,
URA3 marker
Plasmid 2
Stalling sequence
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
pRS315 empty vector,
LEU2 marker
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
pRS315 empty vector,
LEU2 marker
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
pRQC2-RQC2_WT,
LEU2 marker
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
pRQC2-RQC2_hpa
(D9A, D98A, R99A),
LEU2 marker
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
pRS315 empty vector,
LEU2 marker
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
pRQC2-RQC2_WT,
LEU2 marker
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
pRQC2-RQC2_hpa
(D9A, D98A, R99A),
LEU2 marker
cgg cga cga cgg cga cgc cga
cga cga cgg cgc cgc
pRS315 empty vector,
LEU2 marker
2
Strain
yOBj060
yOBj061
yOB542
yOB462
yPS1234
yPS1250
yPS1251
yPS1252
Background
BY4741
ura3::HSEEmGFP
rqc2::kan
rqc1::nat
rps0a::hyg
BY4741
ura3::HSEEmGFP
rqc2::kan
rqc1::nat
rps0a::hyg
BY4741 rqc13xFLAG::kan
ltn1ΔRING::nat
BY4741 rqc13xFLAG::kan
rqc2::nat
BY4741 rqc13xFLAG::kan
vms1::nat rqc2PrA::his5
BY4741 rqc13xFLAG::kan
rqc2::nat
BY4741 rqc13xFLAG::kan
rqc2::nat
BY4741 rqc13xFLAG::kan
rqc2::nat
yOBj142
BY4741
yOBj143
BY4741 ltn1::kan
yOBj144
BY4741 rqc2::kan
yOBj145
BY4741 ltn1::kan
rqc2::nat
yOBj025
yOBj028
Cell lysis
BY4741 ltn1::kan
BY4741 ltn1::kan
rqc2::nat
Plasmid 1
Plasmid 2
Stalling sequence
pRQC2-RQC2_WT,
LEU2 marker
pRQC2-RQC2aaa (D9A,
D98A, R99A), LEU2
marker
pJR3462-VMS1-GFP,
URA3 marker
pRS315 empty vector,
LEU2 marker
pRQC2-RQC2_WT,
LEU2 marker
pRQC2-RQC2_aaa
(D9A, D98A, R99A),
LEU2 marker
pTDH3GFP_TEV_R4_RFP,
URA3 marker
pTDH3GFP_TEV_R4_RFP,
URA3 marker
pTDH3GFP_TEV_R4_RFP,
URA3 marker
pTDH3GFP_TEV_R4_RFP,
URA3 marker
pTDH3-GFP-Rz-His3
pTDH3-GFP-Rz-His3
cga cga cga cga ata aat aaa
ata gat aga
cga cga cga cga ata aat aaa
ata gat aga
cga cga cga cga ata aat aaa
ata gat aga
cga cga cga cga ata aat aaa
ata gat aga
Supplementary Figure 1
Fig S1. Reconstruction statistics of RQC-related structures. Number of particles, surface
views, local resolution heat maps, and FSC plots shown. ‘Gold standard’ FSC cutoffs at 0.143
indicated by dashed lines. Extra-ribosomal densities colored as in Figure 1.
Supplementary Figure 2
Fig S2. 3D classification scheme. Flow chart depicting 3D classification from an initial
consensus structure, revealing distinct classes containing tRNA(s), Tif6p, Rqc2p, and
Ltn1pΔRING.
Supplementary Figure 3
Fig S3. Co-immunoprecipitation and structures of rqc2Δ or Tif6p-containing particles.
(A) Silver stained Ltn1ΔRING and rqc2Δ samples following αFLAG co-immunoprecipitation.
Pertinent protein bands labeled; co-IP of untagged yeast shown to indicate background
contaminant bands. (B) Cryo-EM reconstruction of the rqc2Δ RQC particle with the antiassociation factor Tif6p density indicated. (C) Refined ‘crown view’ structure of the 3D class
containing Tif6p bound to peptidyl-tRNA-60S (from the Rqc1-FLAG, Ltn1ΔRING strain; see
bottom right of Fig. S2). (D) Tif6p crystal structure (orange ribbon, PDB entry 4A18 (40)) fit
within the cryoEM density.
Supplementary Figure 4
Fig S4. Identification of RQC factors by difference mapping. (A) Rqc2p density isolated by
computing the difference between Rqc2p and Rqc2Δ structures. (B) Ltn1pΔRING density
isolated by computing the difference between Rqc2p-classified structures (see bottom left of Fig.
S2).
Supplementary Figure 5
Fig. S5. Ltn1pΔRING structure analysis. (A) Ltn1pΔRING connects with Rqc2p and the 60S
subunit at the sarcin-ricin loop (SRL). Inset specifies view. (B) Structural comparison between
the 60S-bound Ltn1pΔRING and solution structure of Ltn1p (EMD-2252, (6)). Amino-(N) and
carboxy-(C) termini indicated. (C) Structural comparison between the yeast 60S-bound
Ltn1pΔRING and mammalian 60S-bound Listerin (7). L1 = L1 stalk; St = P-stalk
Supplementary Figure 6
Figure S6. RQC binding is incompatible with 80S ribosomes. 40S (light blue) docked over
60S-Rqc2p and 60S-Rqc2p-Ltn1pΔRING structures. Top, surface views. Bottom, cut-away
views. Representative clashes indicated by dotted red line.
Supplementary Figure 7
Fig S7. Comparison of Rqc2p-bound tRNAs with canonical A/A, P/P tRNA states. Top,
Rqc2p-bound tRNAs colored and viewed as in Fig. 2B. Classical A/A (light grey) and P/P (dark
grey) tRNAs superimposed. Bottom, rotated view indicates displacement of elbows and
anticodon loops between classical and Rqc2p-bound tRNAs.
Supplementary Figure 8
Fig S8. Rqc2p-mediated extensions persist in reporters containing CGA repeats or lacking
a stop codon. (A) Immunoblot analysis of a stalling reporter encoding four consecutive CGA
codons. This reporter was also designed to encode STOP codons upon +1 or +2 frame shifting.
(B) Immunoblot analysis of the nonstop GFP-Rz reporter derived from the hammerhead
ribozyme sequence (19). (C) Total amino acid analysis of the R4 stalling reporter, shown in (A).
Also note that frame shifting of this mRNA could not lead to translation of alanine codons.
21
yOBj145
yOBj025
yOBj028
rqc2::nat
BY4741 ltn1::kan
BY4741 ltn1::kan
rqc2::nat
GFP_TEV_R4_RFP,
URA3 marker
pTDH3-GFP-Rz-His3
ata gat aga
pTDH3-GFP-Rz-His3
Cell lysis
Cells were grown in either rich media or synthetic dropout media and harvested at OD600 of
~1.5. The cells were pelleted at 4,500 x g for 6 minutes, washed in ice cold water, pelleted again
at 4,000 x g for 6 minutes, then resuspended in lysis buffer (50mM HEPES-KOH pH 6.8,
150mM KOAc, 2mM Mg(OAc)2, 1mM CaCl2, 0.2M sorbitol, supplemented with cOmplete
protease inhibitor cocktail (Roche). Cell droplets were frozen in liquid nitrogen, lysed into
3
powder using a Freezer/Mill® (SPEX SamplePrep), and stored at -80°C until use.
RQC immunoprecipitation
RQC particles were purified as described (1). Yeast powder was thawed on ice and
resuspended in immunoprecipitation buffer: 100mM KOAc, 10mM MgCl2, 25mM HEPES-KOH
pH 7.4, 0.5mM DTT, supplemented with cOmplete protease inhibitors (Roche). Supernatant was
clarified by centrifugation and incubated with anti-FLAG M2 agarose resin (Sigma) or IgGcoated magnetic Dynabeads (Life Technologies) for 1 hour at 4°C. Resins were washed
thoroughly and the RQC particles were eluted with excess 3xFLAG peptide (Rqc1p-3XFLAG)
or by incubation with HRV 3C protease (Rqc2p-ProteinA) for 1 hour at 4°C. Purified samples
Supplementary Figure 9
Fig S9. CAT tail formation in rqc1Δ cells. Amino acid analysis of GFP-TEV-R12-RFP in
rqc1Δ and rqc1Δrqc2Δ backgrounds. Ala and Thr residues indicated.
Supplementary Figure 10
Fig S10. Edman degradation sequencing of TEV-released CAT tails. Each cycle represents
an average of two replicates and shows the proportion of residues versus the sum of all amino
acids. Note that SGSGSTS is the plasmid-encoded TEV linker upstream of the R12 stall.
Supplementary Figure 11
Fig S11. Sequence and predicted structural alignment between Rqc2p and the NFACT-N
protein Fbp (S. aureus, PDB entry 3DOA). Identical residues shaded gray. Insertion relative to
template shaded burgundy. Deletion relative to template shaded orange. Predicted alpha helices
are colored in green and beta sheets in blue. Deeply conserved residues from the active sites of
some nucleic-acid modifying members of the NFACT family are annotated in red text and
together considered the predicted active site residues. The triple mutant D9A, D98A, R99A = the
rqc2aaa allele. The sequence-to-structure alignment was generated with Phyre2 (41).
Supplementary Figure 12
Fig S12. Rqc2p NFACT-N modeling and RQC IP of rqc2aaa. (A) Rigid-body fits of the
NFACT-N and tandem HhH lobes of the Fbp structure (PDB entry 3DOA, ribbon representation)
docked within the Rqc2p cryoEM density. Rqc2p and the tRNA densities were segmented
automatically with the Segger algorithm as implemented within UCSF Chimera (33, 35). D9,
D98, R99 are shown as space filling models. (B) Silver stained SDS-PAGE of Rqc1p-FLAG IPs
from rqc2Δ backgrounds re-expressed with empty vector (EV), WT RQC2, and rqc2aaa. (C)
Negative stain electron microscopy field of RQC particles purified from rqc2Δ strains harboring
the rqc2aaa plasmid.
25
Supplementary Figure 13
Fig S13. Schematic model for RQC-mediated recognition and rescue of stalled ribosomes.
The 80S ribosome stalls during translation and the 40S dissociates. Ltn1p and Rqc2p recognize
unique features of the resulting peptidyl-tRNA-60S complex. Ltn1p binds Rqc2p and the 60S at
the SRL and reaches around the 60S towards the exit tunnel to ubiquitylate emerging nascent
chains. Cdc48p and its co-adaptors target the ubiquitylated peptide to the proteasome for
degradation. Rqc2p binds the exposed ~P-site tRNA, and directs elongation of the stalled nascent
chain with CAT tails by recruiting tRNAAla(IGC) and tRNAThr(IGU) to the A-site. Rqc2p directed
translation elongation is mRNA template-free and 40S-free. The resulting CAT tail is involved in
signaling to Heat Shock Factor 1, Hsf1p.
Table S1
Nano-ESI-FT-ICR data of trypsin-released CAT tails
CAT tail
peptide length
amino acid
composition
theoretical
mass (Da)
observed
mass (Da)
ppm difference
(observed vs theoretical)
5
4A, 1T
403.2067
403.2071
1.0
6
3A, 3T
534.2649
534.2656
1.3
6
4A, 2T
504.2544
504.2554
2.0
6
5A, 1T
474.2438
474.2445
1.5
7
5A, 2T
575.2915
575.2925
1.7
7
6A, 1T
545.2809
545.2816
1.3
8
6A, 2T
646.3286
646.3294
0.8
9
7A, 2T
717.3657
717.3666
1.3
12
5A, 7T
1080.5299
1080.5310
1.0
12
6A, 6T
1050.5193
1050.5214
2.0
12
7A, 5T
1020.5087
1020.5096
0.9
13
6A, 7T
1151.5670
1151.5696
2.3
13
7A, 6T
1121.5564
1121.5584
1.8
14
8A, 6T
1192.5935
1192.5962
2.3
19
11A, 8T
1607.8002
1607.8036
2.1
protease inhibitor cocktail (Roche). Cell droplets were frozen in liquid nitrogen, lysed into
powder using a Freezer/Mill® (SPEX SamplePrep), and stored at -80°C until use.
RQC immunoprecipitation
RQC particles were purified as described (1). Yeast powder was thawed on ice and
resuspended in immunoprecipitation buffer: 100mM KOAc, 10mM MgCl2, 25mM HEPES-KOH
pH 7.4, 0.5mM DTT, supplemented with cOmplete protease inhibitors (Roche). Supernatant was
clarified by centrifugation and incubated with anti-FLAG M2 agarose resin (Sigma) or IgGcoated magnetic Dynabeads (Life Technologies) for 1 hour at 4°C. Resins were washed
thoroughly and the RQC particles were eluted with excess 3xFLAG peptide (Rqc1p-3XFLAG)
or by incubation with HRV 3C protease (Rqc2p-ProteinA) for 1 hour at 4°C. Purified samples
were verified by silver stain SDS-PAGE and negative stain electron microscopy and used
immediately for cryo-EM experiments.
Electron microscopy
RQC particles suffered from different orientation biases on ultrathin carbon-coated lacey
grids versus when suspended in the holes of Quantifoil holey carbon grids. Thus, we used both
types of grids to obtain a wider distribution of particle views. In brief, 4.5µl of purified RQC
immediately for cryo-EM experiments.
Electron microscopy
RQC particles suffered from different orientation biases on ultrathin carbon-coated lacey
grids versus when suspended in the holes of Quantifoil holey carbon grids. Thus, we used both
types of grids to obtain a wider distribution of particle views. In brief, 4.5µl of purified RQC
were applied to either glow-discharged lacey carbon grids with an ultrathin layer of carbon
deposited on top (Ted Pella) or to chloroform-washed, non-glow-discharged Quantifoil R2/2
grids (SPI Supplies). RQC particles only entered the holes of hydrophobic grids washed with
chloroform. Uniformly thin ice was achieved by manually spreading the sample droplet across
the grid surface using a pipette tip and by applying the sample to both sides of the grid. Grids of
both types were plunge frozen in liquid ethane using a Vitrobot III system (FEI) (40s wait time,
3.0s blot time, operating at 4°C and 75% relative humidity) and stored in liquid nitrogen.
4
The rqc2Δ dataset was collected on an FEI Titan Krios operating at 300kV equipped with a
Falcon I direct electron detector. Images were recorded automatically using EPU (FEI) at a
nominal magnification of 47,000×, which corresponds to a pixel size of 1.37 Å. The dataset
ranged between 0.8 to 2.5µm under focus and each micrograph had a total dose of ~25 electrons
per Å2.
The Rqc1p-3XFLAG and Rqc2p-ProteinA datasets were collected on a Tecnai Polara (FEI)
operating at 300kV, equipped with the K2 Summit direct electron detector (Gatan), as described
(27). Images were recorded using the acquisition program UCSFImage4 (written by X. Li (27)),
with a defocus range from 0.8 to 2.0µm. We recorded movies in super-resolution counting mode
at a magnification of 31,000×, which corresponds to a physical pixel size of 1.22 Å. The beam
intensity was set to ~8 counts per physical pixel per second, corresponding to a dose rate of ~10
electrons per physical pixel per second. Movies were recorded as a stack of 24 subframes, each
of which was accumulated for 0.2 s, resulting in an average of ~1.7 counts per physical pixel in
each subframe and a total specimen dose of ~35 electrons per Å2. All subframes per movie were
motion corrected as implemented within UCSFImage4.
Image processing and 3D reconstruction
CTF parameters for micrographs were determined using the program CTFFIND (28). The
power spectrum for each micrograph was visually inspected and those with poor Thon rings were
each subframe and a total specimen dose of ~35 electrons per Å2. All subframes per movie were
motion corrected as implemented within UCSFImage4.
Image processing and 3D reconstruction
CTF parameters for micrographs were determined using the program CTFFIND (28). The
power spectrum for each micrograph was visually inspected and those with poor Thon rings were
rejected from downstream analysis. Particle images were extracted from micrographs in a semiautomated manner using the swarm tool in e2boxer.py as part of the EMAN2 package (29).
All 2D and 3D classifications and refinements were performed using the RELION package
(30). Particles were sorted into 2D classes, followed by rejecting non-60S particles (i.e.,
incoherent particles and contaminant 80S particles) from downstream analysis. The remaining
particles were then used to compute a consensus 3D reconstruction, after which the aligned
particles were 3D classified in an unsupervised manner. Individual classes were then further
refined until convergence using the auto-refine procedure in RELION. Because particle
alignment is dominated by the larger 60S structure, we further applied a soft spherical mask
around the Rqc2p density in order to improve the quality of the extra-ribosomal features (at the
expense of a lower resolution ribosome). Local resolution of each map was computed using
ResMap (31), using unfiltered map halves as outputted by RELION. Difference maps were
calculated using Bsoft (bop program (32)).
Structure visualization and modeling
Rigid body fittings were done in UCSF Chimera (33). Extra-ribosomal densities were
distinguished by computing difference maps between cryoEM reconstructions and reference
5
expense of a lower resolution ribosome). Local resolution of each map was computed using
ResMap (31), using unfiltered map halves as outputted by RELION. Difference maps were
calculated using Bsoft (bop program (32)).
Structure visualization and modeling
Rigid body fittings were done in UCSF Chimera (33). Extra-ribosomal densities were
distinguished by computing difference maps between cryoEM reconstructions and reference
yeast 60S maps from crystal structure coordinates (PDB entries 3U5D & 3U5E (34)). Individual
components (i.e., Rqc2p, Ltn1p, tRNAs, nascent chains) were isolated using the Segger tools as
implemented within UCSF Chimera (33, 35).
Based on predicted structural conservation between the N-terminal domain of Rqc2p and
other members of the NFACT family of proteins, we first manually guided the fitting of the
NFACT-N domain of the Fbp crystal structure (PDB entry 3DOA, residues 1-140) into the
Rqc2p density, and then used UCSF Chimera to refine the rigid-body fitting. tRNA structures
were obtained from PDB entry 2J00 (36) and fitted as rigid bodies into reconstructions using
UCSF Chimera (33).
TGIRT-seq of RQC-associated RNAs
Total RNA isolated from RQC particles that had been purified from RQC2 and rqc2Δ
strains was analyzed by deep sequencing using a thermostable group II intron reverse
6
Rqc2p density, and then used UCSF Chimera to refine the rigid-body fitting. tRNA structures
were obtained from PDB entry 2J00 (36) and fitted as rigid bodies into reconstructions using
UCSF Chimera (33).
TGIRT-seq of RQC-associated RNAs
Total RNA isolated from RQC particles that had been purified from RQC2 and rqc2Δ
strains was analyzed by deep sequencing using a thermostable group II intron reverse
6
transcriptase (TGIRT) for RNA-seq library construction (9, 37). The TGIRT enhances read
through of modified or edited tRNA bases, facilitating generation of full-length cDNA copies of
tRNAs, and it initiates reverse transcription by a template-switching mechanism that is sensitive
to whether or not the 3’ end of the tRNA is aminoacylated. Total RNAs were extracted from the
purified RQC by using Trizol. Purified RNAs were precipitated with isopropanol, washed in
75% ethanol, and dissolved in water. Half of the sample was deacylated by incubation in 0.1 M
Tris-HCl (pH 9.0) for 45 min at 37°C, as described (38). Portions of the purified RNA samples
before and after deacylation were analyzed with the Small RNA Analysis Kits on a 2100
Bioanalyzer (Agilent Technologies) to assess the relative abundance of tRNAs to 5S rRNA and
5.8S rRNA.
The construction of RNA-seq libraries via TGIRT template switching was done essentially
as described (9, 37). The initial template-primer substrate for template-switching reactions
consists of a 41-nt RNA oligonucleotide (5'-AGA UCG GAA GAG CAC ACG UCU AGU UCU
ACA GUC CGA CGA UC/3SpC3/-3') with Illumina Read1 and Read2 primer binding sites and
a 3' blocking group (three carbon spacer; IDT) annealed to a complementary 42-nt 32P-labeled
Tris-HCl (pH 9.0) for 45 min at 37°C, as described (38). Portions of the purified RNA samples
before and after deacylation were analyzed with the Small RNA Analysis Kits on a 2100
Bioanalyzer (Agilent Technologies) to assess the relative abundance of tRNAs to 5S rRNA and
5.8S rRNA.
The construction of RNA-seq libraries via TGIRT template switching was done essentially
as described (9, 37). The initial template-primer substrate for template-switching reactions
consists of a 41-nt RNA oligonucleotide (5'-AGA UCG GAA GAG CAC ACG UCU AGU UCU
ACA GUC CGA CGA UC/3SpC3/-3') with Illumina Read1 and Read2 primer binding sites and
a 3' blocking group (three carbon spacer; IDT) annealed to a complementary 42-nt 32P-labeled
DNA primer, which leaves an equimolar mixture of A, C, G, or T single-nucleotide 3' overhangs.
Reactions were done with RQC RNA (~100-200 ng), initial template-primer substrate (100 nM),
TGIRT (TeI4c-MRF RT with a C-terminal truncation of the DNA endonuclease domain; 1 µM,
5 units/µl) and 1 mM dNTPs (an equimolar mix of dATP, dCTP, dGTP, and dTTP) in 20 µl of
reaction medium containing 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5, 1 mM DTT.
After pre-incubating a mixture of all components except dNTPs at room temperature for 30 min,
reactions were initiated by adding dNTPs, incubated at 60°C for 30 min, and terminated by
adding 5 M NaOH to a final concentration of 0.25 M, incubating at 95°C for 3 min, and then
7
neutralizing with 5 M HCl. The labeled cDNAs were analyzed by electrophoresis in a denaturing
6% polyacrylamide gel, which was scanned with a Typhoon FLA9500 phosphorImager (GE
Healthcare). Gel regions containing cDNAs with sizes of ~60-nt and ~61-190-nt, including the
42-nt primer added by template-switching, were electroeluted using a D-tube Dialyzer Maxi with
MWCO of 6-8 kDa (EMD Millipore) and ethanol precipitated in the presence of 0.3 M sodium
acetate and linear acrylamide carrier (25 µg; Thermo Scientific). The purified cDNAs were then
circularized with CircLigase II (Epicentre; manufacturer’s protocol with an extended incubation
time of 5 h at 60°C), extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and ethanol
precipitated. The circularized cDNA products were amplified by PCR with Phusion-HF (Thermo
Scientific) using Illumina multiplex (5'- AAT GAT ACG GCG ACC ACC GAG ATC TAC
ACG TTC AGA GTT CTA CAG TCC GAC GAT C -3') and barcode (5'- CAA GCA GAA
GAC GGC ATA CGA GAT BARCODE GTG ACT GGA GTT CAG ACG TGT GCT CTT
CCG ATC T -3') primers under conditions of initial denaturation at 98oC for 5 s, followed by 12
cycles of 98°C for 5 s, 60°C for 10 s and 72°C for 10 s.
Samples were sequenced on an Illumina MiSeq or HiSeq (100-nt single end reads). Read
quality was assessed using FastQC (see
http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were trimmed and filtered to
remove contaminant primer sequences and substandard base calls from the 3' ends using Fastx-
Scientific) using Illumina multiplex (5'- AAT GAT ACG GCG ACC ACC GAG ATC TAC
ACG TTC AGA GTT CTA CAG TCC GAC GAT C -3') and barcode (5'- CAA GCA GAA
GAC GGC ATA CGA GAT BARCODE GTG ACT GGA GTT CAG ACG TGT GCT CTT
CCG ATC T -3') primers under conditions of initial denaturation at 98oC for 5 s, followed by 12
cycles of 98°C for 5 s, 60°C for 10 s and 72°C for 10 s.
Samples were sequenced on an Illumina MiSeq or HiSeq (100-nt single end reads). Read
quality was assessed using FastQC (see
http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were trimmed and filtered to
remove contaminant primer sequences and substandard base calls from the 3' ends using FastxToolkit (see http://hannonlab.cshl.edu/fastx_toolkit) and Ea-utils (http://code.google.com/p/eautils), respectively. Trimmed and filtered reads were aligned to the yeast genome reference
sequence (Ensembl EF4) with Bowtie 2 using end-to-end alignment, yielding 0.5-2 million
(MiSeq) or 2-4 million (HiSeq) mapped reads.
8
Western Blots
Yeast were grown to saturation overnight in SD-URA media, diluted to OD600 0.1 in the
morning, and grown for 3-5 hours to OD600 0.5. The cultures were then normalized to 5ml of
OD600 0.2, pelleted by centrifugation, resuspended in 20µl NuPAGE® LDS Sample Buffer (Life
Technologies), and lysed by boiling for 5 minutes. The samples were centrifuged and 5ul of
supernatant was loaded into NuPAGE® Novex® 4-12% Bis-Tris Precast protein gels (Life
Technologies).
The proteins were transferred onto Novex® nitrocellulose membranes (Life Technologies)
using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). The membranes were blocked in 5%
nonfat milk in TBST for 1 hour, incubated overnight in primary antibodies (αGFP, MA5-15256,
Pierce and α-Hexokinase, H2035-01, US Biological), washed in TBST for 10 minutes, incubated
in DyLight secondary antibodies (Pierce) for 1 hour, and washed 3 times in TBST for 5 minutes
each. The membranes were visualized using the VersaDoc™ system (Bio-Rad).
TEV protease digestion
For the TEV reaction, 4µl of clarified lysate from frozen yeast powder was incubated with
2ul of ProTEV Plus (Promega) with 1mM DTT at room temperature for 2 hours. 6.6µl of
NuPAGE® LDS Sample Buffer (Life Technologies) was added, and the samples were boiled for
supernatant was loaded into NuPAGE® Novex® 4-12% Bis-Tris Precast protein gels (Life
Technologies).
The proteins were transferred onto Novex® nitrocellulose membranes (Life Technologies)
using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). The membranes were blocked in 5%
nonfat milk in TBST for 1 hour, incubated overnight in primary antibodies (αGFP, MA5-15256,
Pierce and α-Hexokinase, H2035-01, US Biological), washed in TBST for 10 minutes, incubated
in DyLight secondary antibodies (Pierce) for 1 hour, and washed 3 times in TBST for 5 minutes
each. The membranes were visualized using the VersaDoc™ system (Bio-Rad).
TEV protease digestion
For the TEV reaction, 4µl of clarified lysate from frozen yeast powder was incubated with
2ul of ProTEV Plus (Promega) with 1mM DTT at room temperature for 2 hours. 6.6µl of
NuPAGE® LDS Sample Buffer (Life Technologies) was added, and the samples were boiled for
5 minutes. The samples were centrifuged and 5µl of supernatant was loaded into NuPAGE®
Novex® 4-12% Bis-Tris Precast protein gels (Life Technologies).
Amino Acid Analysis
Frozen yeast lysate powder was prepared, resuspended, and clarified as described above.
2ml of clarified lysate was incubated with 50µl of GFP-Trap® (Chromotek) for 1 hour at 4°C,
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NuPAGE® LDS Sample Buffer (Life Technologies) was added, and the samples were boiled for
5 minutes. The samples were centrifuged and 5µl of supernatant was loaded into NuPAGE®
Novex® 4-12% Bis-Tris Precast protein gels (Life Technologies).
Amino Acid Analysis
Frozen yeast lysate powder was prepared, resuspended, and clarified as described above.
2ml of clarified lysate was incubated with 50µl of GFP-Trap® (Chromotek) for 1 hour at 4°C,
and washed 3 times in IP buffer for 5 minutes each. The washed GFP-Trap® (Chromotek) resin
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was incubated for 2 minutes in IP buffer adjusted to pH 2.0 in order to acid-elute the samples.
Amino acid analysis was performed by UC Davis Genome Center using the following
protocol, adapted from (39): samples were dried in a glass hydrolysis tube. The samples were
then hydrolyzed in 6N HCl, 1% phenol at 110°C for 24hrs under vacuum. After hydrolysis, the
samples were cooled, unsealed, and dried again. The dried samples were dissolved in 1mL
sodium diluent (Pickering) with 40nmol/mL NorLeucine added as an internal standard. For each
run, 50µl of sample was injected onto the ion-exchange column.
An amino acid standards solution for protein hydrolysate on the Na-based Hitachi 8800
(Sigma, A-9906) is used to determine response factors, and thus calibrate the Hitachi 8800
analyzer for all of the amino acids. In addition this standard has been verified against the
National Institute of Standards and Technology (NIST) standard reference material 2389a.
Edman degradation sequencing
GFP IP was performed as described above. Washed GFP-Trap® (Chromotek) resin was
incubated with ProTEV Plus (Promega) with 1mM DTT overnight at 4°C. The supernatant was
analyzer for all of the amino acids. In addition this standard has been verified against the
National Institute of Standards and Technology (NIST) standard reference material 2389a.
Edman degradation sequencing
GFP IP was performed as described above. Washed GFP-Trap® (Chromotek) resin was
incubated with ProTEV Plus (Promega) with 1mM DTT overnight at 4°C. The supernatant was
collected and added to NuPAGE® LDS Sample Buffer (Life Technologies). Samples were
boiled for 5 minutes, run on a NuPAGE® Novex® 12% Bis-Tris Precast gel (Life
Technologies), and transferred to a PVDF membrane (Bio-Rad). The membrane was visualized
with Coomassie blue staining. The low molecular weight band that ran at the gel front was
excised and subject to Edman sequencing at Stanford University’s Protein and Nucleic Acid
Facility.
N-terminal sequence analysis is determined using Edman chemistry on an Applied
Biosystems (AB) Procise liquid-pulse protein sequenator. PTH-Amino Acids are separated on a
Brownlee C-18 reverse phase column (2.1 mm x 22 cm) at 55 C using a linear gradient of 1410
to
44% buffer B over 17.6 min. Buffer A is 3.5% tetrahydrofuran and 2% AB premix buffer
concentrate. (The recipe for the concentrate is proprietary to AB, but they have published the
approximate composition as follows: 10-20% Acetic Acid, < 10% Sodium Acetate, < 10%
Sodium Hexanesulfonate, 65-75% Water.) Buffer B is 12% Isopropanol in Acetonitrile. PTHamino acid analysis is performed by Model 610A software, version 2.1.
GFP Fluorescence Measurements
Fluorescence was measured using a LSRII flow cytometer (BD). All fluorescent
Brownlee C-18 reverse phase column (2.1 mm x 22 cm) at 55 C using a linear gradient of 14 to
44% buffer B over 17.6 min. Buffer A is 3.5% tetrahydrofuran and 2% AB premix buffer
concentrate. (The recipe for the concentrate is proprietary to AB, but they have published the
approximate composition as follows: 10-20% Acetic Acid, < 10% Sodium Acetate, < 10%
Sodium Hexanesulfonate, 65-75% Water.) Buffer B is 12% Isopropanol in Acetonitrile. PTHamino acid analysis is performed by Model 610A software, version 2.1.
GFP Fluorescence Measurements
Fluorescence was measured using a LSRII flow cytometer (BD). All fluorescent
measurements were performed at 25˚C at log phase in synthetic complete media (SC). Matlab
7.8.0 (Mathworks) was then used to compute the median internally-normalized values
(GFP/sidescatter for the HSF reporter). Values were then normalized to WT and log2 scores
computed, so that the wild-type strain had value 0. Error bars are standard deviations between
technical replicates.
Trypsin Digestion
TEV-released CAT tails in solution were digested with TPCK-modified trypsin. Trypsin (in
50 mM NH4HCO3) was added to the solution, adjusted to pH 7.9 with dilute NH4OH to obtain a
ratio of approximately 1 to 25 (enzyme to protein). Digest reactions were allowed to continue
over night at 37°C. Alternatively, gel bands of interest were excised, de-stained twice in 1 ml
volumes of 50 mM NH4HCO3 in 50% methanol for 1 hour with gently mixing. Gel bands were
then rehydrated in 1 ml of 50 mM NH4HCO3 for 30 min, cut into several pieces then dehydrated
in 1 ml of 100% acetonitrile for 30 min at room temperature with gentle shaking. Acetonitrile
computed, so that the wild-type strain had value 0. Error bars are standard deviations between
technical replicates.
Trypsin Digestion
TEV-released CAT tails in solution were digested with TPCK-modified trypsin. Trypsin (in
50 mM NH4HCO3) was added to the solution, adjusted to pH 7.9 with dilute NH4OH to obtain a
ratio of approximately 1 to 25 (enzyme to protein). Digest reactions were allowed to continue
over night at 37°C. Alternatively, gel bands of interest were excised, de-stained twice in 1 ml
volumes of 50 mM NH4HCO3 in 50% methanol for 1 hour with gently mixing. Gel bands were
then rehydrated in 1 ml of 50 mM NH4HCO3 for 30 min, cut into several pieces then dehydrated
in 1 ml of 100% acetonitrile for 30 min at room temperature with gentle shaking. Acetonitrile
was removed followed by the addition of 10 to 20 µl of sequence-grade modified trypsin
(Promega) (20 ng/µL) in 50 mM NH4HCO3 and the gel pieces were allowed to swell for 15
minutes on ice. The excess trypsin solution was removed and the same 20 µl buffer (minus
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trypsin) was added to just cover the gel pieces, then incubated overnight at 37°C. The digestion
was quenched by 20 µL of 1% formic acid, allowed to stand, and the supernatant collected.
Further peptide extraction from the gel material was performed twice by the addition of 50%
acetonitrile with 1% formic acid followed by sonication at 37°C for 20 minutes. A final complete
dehydration of the gel pieces was accomplished by addition of 20 µL of 100% acetonitrile and
incubation at 37°C for 20 minutes. All the extracts were pooled, dried and reconstituted in 100
µL of 5% acetonitrile with 0.1% formic acid for LC/MS/MS analysis.
Mass Spectrometry
dehydration of the gel pieces was accomplished by addition of 20 µL of 100% acetonitrile and
incubation at 37°C for 20 minutes. All the extracts were pooled, dried and reconstituted in 100
µL of 5% acetonitrile with 0.1% formic acid for LC/MS/MS analysis.
Mass Spectrometry
Trypsin-digested peptides were analyzed using a nano-LC/MS/MS system comprised of a
nano-LC pump (2D-ultra, Eksigent) and a LTQ-FT-ICR mass spectrometer (ThermoElectron
Corporation, San Jose, CA). The LTQ-FT-ICR was equipped with a nanospray ion source
(ThermoElectron Corp.).
Approximately 5 µL of tryptic digest samples were injected onto a dC18 nanobore LC
column. The nanobore column was made in house using dC18 (Atlantis, Waters Corp); 3 µm
particle; column: 75 µm ID x 100 mm length. A linear gradient LC profile was used to separate
and elute peptides with a constant total flow rate of 350 nL/minute. The gradient consisted of 5
to 96% solvent B in 78 minutes (solvent B: 100% acetonitrile with 0.1% formic acid; solvent A:
100% water with 0.1% formic acid) was used for the analyses.
The LTQ-FT-ICR mass spectrometer was operated in the data-dependent acquisition mode
with the “top 10” most intense peaks observed in an FT primary scan selected for on-the-fly
MS/MS. Each peak is subsequently trapped for MS/MS analysis and peptide fragmentation in the
LTQ linear ion trap portion of the instrument. Spectra in the FT-ICR were acquired from m/z
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350 to 1400 Da at 50,000 resolving power. Mass accuracies are typically within about 3 ppm.
The LTQ linear ion trap was operated with the following parameters: precursor activation time
30 ms and activation Q at 0.25; collision energy was set at 35%; dynamic exclusion width was
set at low mass of 0.1 Da and high mass at 2.1 Da with one repeat count and duration of 10 s.
Mascot Database Searches
LTQ-FT/LC/MS/MS raw data files were processed with proteome discoverer 1.4 software
(ThermoElectron Corp., San Jose, CA). Resulting DTA files from each data acquisition file
were searched to identify CAT peptides against the custom databases using the MASCOT search
engine (Matrix Science Ltd.; version 2.2.7; in-house licensed). Searches were done with nonspecificity, allowing two missed cleavages, and a mass error tolerance of 7 ppm in MS spectra
(i.e. FT-ICR data) and 0.7 Da for MS/MS ions. Identified peptides were generally accepted
when the MASCOT ion score value exceeded 20. All the peptide assignments were also
manually validated.