RESEA
RCH
ARTICL
E
Light-dependent
N-end
rule-mediated
disruption
of
protein
function
in
Saccharomyces
cerevisiae
and
Drosophila
melanogaster
Leslie
M.
Stevens
ID
1
☯
, Goheun
Kim
ID
1
☯
, Theodora
Koromila
ID
2
☯
, John
W.
Steele
ID
1
,
James
McGehee
ID
2
, Angelike
Stathopoulos
ID
2
*
, David
S.
Stein
ID
1
*
1
Department
of Molecular
Bioscience
s and
Institute
for
Molecular
and
Cellular
Biology,
The
University
of
Texas
at Austin,
Austin,
Texas,
United
States
of America,
2
Division
of Biology
and
Biological
Engineeri
ng,
Californi
a Institute
of Technolo
gy,
Pasade
na,
California,
United
States
of America
☯
These
authors
contribu
ted
equally
to this
work.
*
angelike
@caltech.
edu
(AS);
d.stein@
mail.utexas.ed
u (DSS)
Abstract
Here
we
describe
the
development
and
characterization
of the
photo-N-degron
, a peptide
tag
that
can
be
used
in optogenetic
studies
of protein
function
in vivo
.
The
photo-N-degron
can
be
expressed
as
a genetic
fusion
to the
amino
termini
of other
proteins,
where
it under-
goes
a blue
light-depend
ent
conformational
change
that
exposes
a signal
for
the
class
of
ubiquitin
ligases,
the
N-recognins,
which
mediate
the
N-end
rule
mechanism
of proteasomal
degradation.
We
demonstrate
that
the
photo-N-degron
can
be
used
to direct
light-mediated
degradation
of proteins
in
Saccharomyces
cerevisiae
and
Drosophila
melanogaster
with
fine
temporal
control.
In addition,
we
compare
the
effectiveness
of the
photo-N-degron
with
that
of two
other
light-dependent
degrons
that
have
been
developed
in their
abilities
to medi-
ate
the
loss
of function
of Cactus,
a component
of the
dorsal-ventral
patterning
system
in
the
Drosophila
embryo.
We
find
that
like
the
photo-N-degron
, the
blue
light-inducible
degra-
dation
(B-LID)
domain,
a light-activated
degron
that
must
be
placed
at the
carboxy
terminus
of targeted
proteins,
is also
effective
in eliciting
light-dependent
loss
of Cactus
function,
as
determined
by
embryonic
dorsal-ventral
patterning
phenotypes.
In contrast,
another
previ-
ously
described
photosensitiv
e degron
(psd),
which
also
must
be
located
at the
carboxy
ter-
minus
of associated
proteins,
has
little
effect
on
Cactus-depend
ent
phenotypes
in response
to illumination
of developing
embryos.
These
and
other
observations
indicate
that
care
must
be
taken
in the
selection
and
application
of light-dependent
and
other
inducible
degrons
for
use
in studies
of protein
function
in vivo
,
but
importantly
demonstrate
that
N-
and
C-terminal
fusions
to the
photo-N-degron
and
the
B-LID
domain,
respective
ly,
support
light-dependent
degradation
in vivo
.
PLOS
Genetics
| https://
doi.org/10.13
71/journal.p
gen.100
9544
May
17,
2021
1 / 38
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OPEN
ACCESS
Citation:
Stevens
LM, Kim G, Koromila
T, Steele
JW, McGehee
J, Stathopoulos
A, et al. (2021)
Light-depen
dent N-end
rule-mediate
d disruptio
n of
protein
function
in
Saccharom
yces cerevisiae
and
Drosophil
a melanoga
ster
. PLoS
Genet
17(5):
e1009544.
https://d
oi.org/10.1371/j
ournal.
pgen.100954
4
Editor:
R. Scott
Hawley,
Stowers
Institute
for
Medical
Research,
UNITED
STATES
Received:
October
8, 2020
Accepted:
April 12, 2021
Published:
May 17, 2021
Peer Review
History:
PLOS
recognize
s the
benefits
of transpar
ency
in the peer review
process;
therefore,
we enable
the publication
of
all of the content
of peer review
and author
response
s alongside
final,
published
articles.
The
editorial
history
of this article
is available
here:
https://doi.o
rg/10.1371/jo
urnal.pgen.1
009544
Copyright:
©
2021
Stevens
et al. This is an open
access
article
distributed
under
the terms
of the
Creative
Commons
Attribution
License,
which
permits
unrestricte
d use, distribu
tion, and
reproduction
in any medium,
provided
the original
author
and source
are credited.
Data
Availabilit
y Statement:
All relevant
data are
within
the manuscript
and its Support
ing
Information
files.
Author
summary
Much
of
what
we
know
about
biological
processes
has
come
from
the
analysis
of
mutants
whose
loss-of-function
phenotypes
provide
insight
into
their
normal
functions.
However,
for
genes
that
are
required
for
viability
and
which
have
multiple
functions
in
the
life
of
a
cell
or
organism
one
can
only
observe
mutant
phenotypes
produced
up
to
the
time
of
death.
Normal
functions
performed
in
wild-type
individuals
later
than
the
time
of
death
of
mutants
cannot
be
observed.
In
one
approach
to
overcoming
this
limitation,
a class
of
peptide
degradation
signals
(degrons)
have
been
developed,
which
when
fused
to
pro-
teins-of-interest,
can
target
those
proteins
for
degradation
in
response
to
various
stimuli
(temperature,
chemical
agents,
co-expressed
proteins,
or
light).
Here
we
describe
a new
inducible
degron
(the
photo-N-degron
or
PND),
which
when
fused
to
the
N-terminus
of
a protein,
can
induce
N-end
rule-mediated
degradation
in
response
to
blue-light
illumina-
tion
and
have
validated
its
use
in
both
yeast
and
Drosophila
embryos.
Moreover,
using
the
Drosophila
embryonic
patterning
protein
Cactus,
we
show
that
like
the
PND,
the
previ-
ously-described
B-LID
domain,
but
not
the
previously-described
photosensitive
degron
(psd),
can
produce
detectable
light-inducible
phenotypes
in
Drosophila
embryos
that
are
consistent
with
the
role
of
Cactus
in
dorsal-ventral
patterning.
Introduction
More
than
a century
of
genetic
analysis
underlies
much
of
our
understanding
of
biology.
Unbiased
genetic
screens
utilizing
chemical
mutagens,
ionizing
radiation
or
insertional
muta-
genesis
with
transposons
or
retroviruses,
and
more
recently,
reverse
genetic
strategies
capable
of
generating
precisely
targeted
mutations,
have
been
critical
in
uncovering
the
genes,
proteins
and
mechanisms
underlying
normal
physiology
as
well
as
the
processes
that
go
awry
in
various
disease
states.
However,
for
genes
with
products
that
are
required
early
in
the
life
of
an
organ-
ism,
it can
be
challenging
to
generate
loss-of-function
mutant
individuals
in
which
later
phe-
notypes
associated
with
protein
loss
can
be
examined.
While
the
use
of
site-specific
recombination
systems
to
generate
clones
of
cells
lacking
expression
of
a protein
in
the
back-
ground
of
an
otherwise
viable
individual
[1–4]
can,
in
some
cases,
overcome
this
barrier,
pro-
teins
already
present
may
perdure
for
some
time
and
even
multiple
cell
generations
after
mutant
clone
induction,
which
can
complicate
the
analysis
of
the
loss-of-function
phenotypes.
This
is especially
problematic
in
situations
in
which
it is desirable
to
achieve
rapid
protein
inactivation,
such
as
investigations
of
protein
function
at
specific
stages
of
the
cell
cycle,
during
cell
migration
and
morphogenesis,
or
during
neuronal
signaling.
Moreover,
for
genes
encod-
ing
proteins
that
are
necessary
for
cell
viability,
cell
death
following
the
generation
of
mutant
clones
can
obscure
the
detection
and
analysis
of
more
subtle
phenotypes
resulting
from
pro-
tein
loss-of-function.
Similarly,
RNA
interference
via
the
expression
of
dsRNA
or
siRNAs,
which
has
been
used
to
interrogate
the
function
of
vital
genes
in
a cellular
or
tissue-specific
manner
[5,6]
often
achieves
only
partial
elimination
of
the
protein-of-interest
and
is also
sus-
ceptible
to
the
problem
of
protein
perdurance
noted
above.
Another
approach
to
the
study
of
proteins
with
functions
essential
for
organismal
or
cell
viability
is the
use
of
temperature-sensit
ive
(TS)
mutations.
However,
although
methods
for
the
rational
design
of
TS
alleles
encoding
proteins-of-interest
exist
[7–10],
these
approaches
are
associated
with
drawbacks
that
can
limit
their
general
applicability.
Dohmen
et
al.
[11]
devised
a general
approach
for
expressing
TS
proteins,
relying
upon
the
N-end
rule
pathway
for
ubiquitin/proteasome
-mediated
degradation
[12–14],
which
degrades
proteins
bearing
N-
PLOS GENE
TICS
A light-depe
ndent
protein
degron
that
functions
in yeast
and
fly
PLOS
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2021
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Funding:
This work
was funded
by grants
from
the
NIH Office
of the Director
(https://w
ww.nih.gov/
institutes-ni
h/nih-office
-director)
R21OD017964
(awarded
to D.S.S.,
supporting
L.M.S.,
G.K.,
and D.
S.S.),
the National
Institute
of General
Medica
l
Sciences
(https://w
ww.nigms.
nih.gov)
R35GM11
8146
(awarded
to A.S., supporting
T.K.
and A.S.),
and the Eunice
Kennedy
Shriver
Nationa
l
Institute
of Child
Health
and Human
Developm
ent
(https://w
ww.nichd.ni
h.gov)
R21HD095639
(awarded
to A.S., supporting
J.M. and A.S.)
of the
National
Institutes
of Health.
J.W.S.
was funded
by
the Cell and Molecula
r Biology
Graduate
Program
and the Institute
for Cellular
and Molecular
Biology
at the University
of Texas
at Austin.
The funders
had no role in study
design,
data collecti
on and
analysis,
decision
to publish,
or prepara
tion of the
manuscript.
Competing
interests
:
The authors
have declared
that no competing
interests
exist.
terminal
amino
acid
residues
other
than
methionine
under
the
control
of
ubiquitin
ligases
known
as
UBRs
or
N-recognins
[15,16].
Dohmen
et
al.
[11]
showed
that
a peptide
comprising
a TS
version
of
the
mouse
dihydrofolate
reductase
protein
carrying
an
N-terminal
arginine
(the
temperature
dependent
or
“td”
degron)
could,
when
fused
to
the
amino
terminus
of
a sev-
eral
yeast
proteins,
render
the
resulting
fusion
proteins
inactive
and
lead
to
their
degradation
at
37 ̊
C but
not
at
23 ̊
C.
This
TS
phenotype
was
dependent
upon
UBR1,
the
yeast
N-recognin
[12,15,17].
The
td
degron
has
been
used
to
investigate
protein
function
in
a number
of
systems
including
budding
yeast,
fission
yeast
and
vertebrate
tissue
culture
cells
[18–23].
Many
organ-
isms
cannot
survive
at
37 ̊
C,
the
temperature
at
which
the
td
degron
mediates
protein
degra-
dation.
Accordingly,
the
low
temperature-controlled
(lt)
degron
[24],
which
operates
over
a
lower
temperature
range
(16 ̊C
to
29 ̊C),
was
generated
by
modifying
the
td
degron,
which
expands
the
organisms
in
which
this
method
can
be
applied.
However,
TS
mutants,
including
the
td
and
lt degrons
cannot
be
used
to
study
protein
function
in
homeothermic
organisms
such
as
mammals,
which
maintain
a constant
internal
temperature.
A number
of
other
degrons
have
been
developed,
which
induce
the
proteins
to
which
they
have
been
attached
to
undergo
degradation
in
response
to
addition
of
small
molecules
such
as
the
rapamycin
analogue
Shield-1
[25,26]
or
auxin
[27–29].
These
approaches
are
highly
depen-
dent
upon
the
extent
and
rapidity
with
which
the
small
molecule
can
be
administered
to
or
depleted
from
the
target
cell/tissue/organism.
Furthermore,
the
auxin
system
as
well
as
several
other
inducible
degron
systems
require
the
co-expression
of
a heterologous
specificity-confer-
ring
factor
together
with
the
degron-tagged
target
protein
[30–33].
While
these
approaches
can
provide
tissue
specificity,
based
on
the
expression
pattern
of
the
specificity
conferring
fac-
tors,
they
are
obviously
influenced
and
potentially
limited
in
utility
by
the
time
required
for
induction
and
expression
or
loss
of
these
factors.
In
recent
years,
a number
of
novel
strategies
have
been
developed
in
which
light
is
employed
to
modulate
protein
behavior
in
powerful
new
approaches
to
examining
biological
processes.
These
new
technologies,
which
comprise
the
rapidly
expanding
area
of
optogenetics
[34,35],
have
revolutionized
several
realms
of
biomedical
research,
leading
to
the
expression
of
light-sensitive
membrane
channels
[36–38]
and
the
generation
of
proteins
that
undergo
light-
dependent
conformational
changes
that
affect
their
activity
[39–41],
cellular
localization
[42–
44],
and
protein-protein
interactions
[45,46].
We
began
our
studies
intending
to
combine
recent
advances
in
the
understanding
of
light-
modulated
proteins
and
of
ubiquitin/proteasome-me
diated
protein
degradation
to
develop
techniques
that
permit
light-activated
degradation
of
target
proteins.
In
addition
to
overcom-
ing
the
limitations
associated
with
the
conditional
approaches
described
above,
such
a system
could
potentially
enable
a level
of
temporal
and
spatial
precision
not
possible
using
currently
available
systems
for
perturbing
gene
expression.
Many
of
the
gene
products
required
for
cor-
rect
embryonic
development
in
Drosophila
are
expressed
maternally
and
deposited
as
mRNAs
or
proteins
into
the
developing
egg
during
oogenesis,
for
later
function
in
the
embryo.
For
those
gene
products
required
for
viability
of
the
female
fly,
or
during
oogenesis
to
produce
an
egg,
it is difficult
to
examine
the
phenotypic
consequences
of
loss
of
function
of
proteins
pro-
duced
maternally
that
are
required
to
support
progeny
embryogenesis.
Thus,
the
ability
to
rap-
idly
eliminate
otherwise
stable
proteins
with
the
application
of
light
could
provide
a valuable
tool
for
the
examination
of
loss-of-function
phenotypes
whose
visualization
would
be
pre-
cluded
by
the
perturbation
of
earlier
loss-of-function
phenotypes.
Proteins
containing
LOV
domains
respond
to
environmental
stimuli
such
as
Light,
Oxygen
and
Voltage
by
undergoing
conformational
changes
[47,48].
Light-dependent
members
of
this
class
of
proteins
utilize
flavin
cofactors
as
chromophores
to
function
as
blue-light
sensitive
photoreceptors
in
bacteria
[49],
fungi
[50,51]
and
plants
[52].
Structural
studies
of
one
of
the
PLOS GENE
TICS
A light-depe
ndent
protein
degron
that
functions
in yeast
and
fly
PLOS
Genetics
| https://
doi.org/10.13
71/journal.p
gen.100
9544
May
17,
2021
3 / 38
two
LOV
domains
present
in
phototropin
1 of
Avena sativa
(common
oat)
(phLOV2)
showed
that
under
blue
light
illumination,
the
flavin-binding
region
dissociates
from
and
unwinds
an
adjacent
alpha
helical
region
termed
J
α
[53–55].
Other
studies
have
recently
established
that
proteins
bearing
modified
LOV
domains
can
be
induced
to
degrade
in
response
to
light
[56,57].
Here
we
report
on
the
development
of
an
additional
light-dependent
degron
that
makes
use
of
the
phLOV2
domain
from
Avena sativa
(oat),
which
we
term
the
photo-N-degron
(PND).
When
attached
to
the
N-terminus
of
several
proteins
which
are
then
expressed
in
yeast,
the
PND
induces
light-dependent
N-end
rule-mediated
loss
of
function,
owing
to
pro-
tein
degradation.
We
also
show
that
in
Drosophila
embryos,
the
PND
can
induce
a rapid
light-
dependent
loss
of
Cactus,
the
cognate
inhibitor
of
Dorsal,
the
fly
orthologue
of
the
mammalian
transcription
factor
NF
κ
B
[58].
Cactus
and
Dorsal
are
components
of
the
signal
transduction
pathway
that
defines
Drosophila
embryonic
dorsal-ventral
(DV)
polarity
[59]
and
light-
induced
degradation
of
PND-tagged
Cactus
leads
to
alterations
in
DV
patterning.
Finally,
we
compare
the
abilities
of
the
psd
[56],
the
B-LID
domain
[57],
and
the
PND,
finding
that,
like
the
PND,
the
B-LID
domain
directs
robust
elimination
of
Cactus
function,
while
the
psd
degron
leads
to
only
a subtle
phenotypic
difference
upon
illumination.
Our
results
demon-
strate
that
the
PND
can
be
a powerful
tool
for
conditional
elimination
of
proteins-of-interest
for
phenotypic
studies
in vivo
and
stress
the
need
for
care
in
the
selection
of
engineered
degrons
for
use
in
studies
of
protein
function.
Results
The
LOV2
domain
from
plant
phototropin
1 bearing
an
N-end
rule
targeted
arginine
domain
directs
light
dependent
loss-of-function
phenotypes
in
yeast
a.
Ura3p.
As
noted
above,
proteins
carrying
LOV-sensitive
domains
respond
to
various
environmental
stimuli
by
undergoing
conformational
changes
[47,48].
We
reasoned
that
if
properly
positioned
at
the
amino
terminus
of
a protein-of-interest,
light-dependent
unwinding
of
the
J
α
helix
within
the
LOV
domain
could
act
analogously
to
the
temperature-dependent
unfolding
of
DHFR
ts
to
facilitate
degradation
of
the
fusion
protein
by
the
N-end
rule
degrada-
tion
pathway.
Proteins
with
atypical
N-terminal
amino
acid
can
be
generated
experimentally
by
expressing
the
protein-of-interest
as
in-frame
fusions
to
the
C-terminus
of
ubiquitin.
Because
the
ubiquitin
monomer
is cleaved
co-translationally
through
the
action
of
a deubiqui-
tinating
enzyme
[11,12,60],
it does
not
mark
the
protein
for
proteasomal
degradation
and
the
amino
acid
immediately
following
the
ubiquitin
becomes
the
N-terminal
residue
of
the
fully
translated
protein.
Accordingly,
we
engineered
constructs
that
would
generate
a protein
bearing
an
N-termi-
nal
ubiquitin
moiety
followed
by
an
arginine
residue
(R),
which
would
correspond
to
the
amino
terminus
after
co-translational
removal
of
ubiquitin.
The
arginine
was
followed
by
the
144
amino
acid
LOV2
domain
of
phototropin
1 from
Avena sativa
(phLOV2),
a single
in-
frame
copy
of
the
HA
epitope
[61]
and
finally
the
coding
sequence
of
the
yeast
orotidine-5’-
phosphate
decarboxylase
protein,
Ura3p,
which
is encoded
by
the
URA3
gene.
The
basic
struc-
ture
of
this
Ubi-R-phLOV2-HA-Ura3p
fusion
protein
and
how
it is presumed
to
direct
light-
inducible
degradation
of
Ura3p
is depicted
in
Fig
1A
and
1L,
respectively.
Because
it was
not
known
whether
the
addition
of
the
R-phLOV2
element
would
render
the
fusion
proteins
too
labile
or
too
stable
to
detect
phenotypic
differences
under
dark
versus
illuminated
conditions,
we
constructed
additional
plasmids
to
express
versions
of
the
protein
in
which
putative
stabi-
lizing
or
destabilizing
stretches
of
amino
acids,
from
DHFR
and
DHFR
ts
, respectively
[11],
PLOS GENE
TICS
A light-depe
ndent
protein
degron
that
functions
in yeast
and
fly
PLOS
Genetics
| https://
doi.org/10.13
71/journal.p
gen.100
9544
May
17,
2021
4 / 38
Fig
1.
An
amino
terminal
domain
encoding
ubiquiti
n,
fused
to
the
LOV2
domain
from
oat
phototr
opin
I
mediates
blue
light/N-
end
rule-me
diated
loss
of
Ura3p
function
in
yeast.
(A)
A schemati
c diagram
showing
the
organizati
on
of
the
construct
encoding
blue/li
ght,
N-end
rule
targeted
Ura3p,
under
the
transcript
ional
control
of
the
copper-i
nducible
CUP1
promoter
(
P
CUP1
). From
5’ to
3’,
the
transgene
encodes
a single
copy
of
the
ubiquitin
open
reading
frame
(
UBI
),
the
LOV2
domain
from
plant
phototropin
I (
LOV
),
a single
copy
of
the
influenza
hemaggluti
nin
epitope
(
HA
),
and
the
open
reading
frame
encoding
the
yeast
Ura3p
protein
(
URA3
).
Protein
synthesis
initiates
at
the
ubiquitin
initiation
codon
(M)
and
the
pair
of
glycine
residues
at
the
C-termi
nus
of
the
ubiquit
in
open
reading
frame
(GG)
are
followed
immediate
ly
by
an
arginine
codon
(R).
The
ubiquitin
domain
is removed
co-translati
onally,
leaving
the
arginine
residue
immediate
ly
preceding
the
LOV
domain
as
the
N-terminal
residue
of
the
mature
protein.
In
the
correspond
ing
UBI-R-DHFR-HA-URA3
construct,
the
sequenc
e encoding
the
LOV2
domain
have
been
replaced
by
PLOS GENE
TICS
A light-depe
ndent
protein
degron
that
functions
in yeast
and
fly
PLOS
Genetics
| https://
doi.org/10.13
71/journal.p
gen.100
9544
May
17,
2021
5 / 38