UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN

Article abstract of DOI:10.1074/jbc.M116.773200

Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric re

Full text of DOI:10.1074/jbc.M116.773200

JBC Papers in Press. Published on July 14, 2017 as Manuscript M116.773200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.773200
UbMES and UbFluor: Novel Probes For RBR E3
Ubiquitin Ligase PARKIN
Sungjin Park1, Peter K. Foote2, David T. Krist2, Sarah E. Rice1, Alexander V. Statsyuk,2,3
1Department of Cell and Molecular Biology, Northwestern University, 303 East Chicago Avenue,
2
Chicago, IL 60611, USA; Chemistry of Life Processes Institute, Department of Chemistry,
Northwestern University, Silverman Hall, 2145 Sheridan Road, Evanston, Illinois 60208, United States;
3Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of
Houston, 3455 Cullen Blvd., Houston, TX 77204-5037.
KEYWORDS PARKIN, Ubiquitin, PINK1, Mitochondria, Miro
Abstract
Ring-Between-Ring (RBR) E3 ligases have
been implicated in autoimmune disorders
and neurodegenerative diseases. The
functions of many RBR E3s are poorly
defined and their regulation is complex,
involving posttranslational modifications and
allosteric regulation with other protein
lysine selection, and polyubiquitin chain
formation. Next, we confirmed that UbFluor
quantitatively detects naturally-occurring
activation states of PARKIN caused by
Ser65-phosphorylation
(pPARKIN)
and
phosphorylated ubiquitin (pUb). Secondly,
we showed that both pUb and the ubiquitin-
accepting substrate contribute to maximal
pPARKIN ubiquitin conjugation turnover.
pUb enhances transthiolation step, while
the substrate clears the pPARKIN~Ub
partners.
The functional complexity of
RBRs, coupled with the complexity of the
native ubiquitination reaction that requires
ATP, E1, and E2 enzymes, makes it difficult
to study these ligases for basic research
and therapeutic purposes. To address this
challenge, we developed novel chemical
probes, ubiquitin C-terminal fluorescein
thioesters UbMES and UbFluor, to
qualitatively and quantitatively assess the
activity of the RBR E3 ligase PARKIN in a
simple experimental set up and in real-time
using fluorescence polarization. We first
confirmed that PARKIN does not require an
E2 enzyme for substrate ubiquitination,
thioester
intermediate.
Lastly,
we
established that UbFluor can quantify
activation or inhibition of PARKIN by
structural
mutations.
These
results
demonstrate the feasibility of using UbFluor
for quantitative studies of the biochemistry
of RBR E3s and for high-throughput
screening of small molecule activators or
inhibitors of PARKIN and other RBR E3
ligases.
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Introduction
was shown that overexpression of PARKIN
in rat and mouse PD models is
neuroprotective, supporting this hypothesis
(34-37). However, while pharmacological
activators of PINK1 are known (38),
activators of ubiquitin ligase PARKIN are
not known in the literature, and attempts to
develop them were not successful (39). To
develop such probes, it is essential to
understand Parkin enzymatic mechanisms.
Parkinson’s disease (PD) is the second
most common type of neurodegenerative
disease, and is characterized by defects in
mitochondrial function. Ubiquitin ligase
PARKIN and PTEN-induced putative
Ser/Thr kinase 1 (PINK1) reconstitute a
major pathway to eliminate dysfunctional
mitochondria and are thought to protect
dopaminergic neurons from oxidative stress
(1). PINK1 acts upstream of PARKIN and is
continuously imported to the mitochondria
where it is rapidly degraded. Upon
mitochondrial damage PINK1 degradation
is inhibited, and PINK1 accumulates at the
mitochondrial outer membrane (MOM) (2-
4). There PINK1 promotes the recruitment
of cytoplasmic PARKIN and activation of its
ubiquitin ligase activity (4). Active PARKIN
causes extensive polyubiquitination of MOM
proteins and triggers mitophagy (5).
PARKIN ubiquitination is antagonized by
PARKIN is a Ring-Between-Ring
(RBR) E3 ligase, which forms an obligatory
RBR E3~Ub thioester intermediate prior to
the transfer of the ubiquitin onto the
acceptor lysine (40-43). PARKIN is
composed of six distinct domains: an N-
terminal ubiquitin-like domain (Ubl), a
unique PARKIN-specific domain (UPD, also
referred to as RING0), RING1, in-between-
RING (IBR), repressor element (REP), and
catalytic RING2 domains (Figure S1E) (43-
45). RING1 domain of PARKIN is thought to
recruit E2~Ub thioester (46), while the
RING2 domain of PARKIN harbours the
catalytic cysteine (40). Mutation of this
catalytic cysteine (C431F) is found in some
Parkinson disease patients (47). Recently it
was shown RING2 domain of RBR E3
HHARI also harbours a weak Ub binding
deubiquitinases (DUBs), such that
a
threshold amount of ubiquitination in a
given time may be necessary to
successfully signal mitophagy (6).
The
mechanism
of
PARKIN
activation is complex, but it is known that
phosphorylation of Ser65 in PARKIN Ubl
domain and Ser65 in mono- and/or poly
ubiquitin chains by PINK1 promote the
mitochondrial translocation and activity of
PARKIN which in turn stimulate mitophagy
(7-22). Dozens of mutations in both PINK1
and PARKIN have been found in patients
site
important
for
E2~Ub/HHARI
transthiolation (48), while RING1-IBR-Ubl
domains of PARKIN form another ubiquitin
binding surface important for PARKIN
activity.
A shared feature of PARKIN and
several other RBR ligases is autoinhibition,
which is relieved by binding partners and/or
by phosphorylation (49-53). In autoinhibited
PARKIN, the E2~Ub binding site on RING1
is blocked by the REP domain, while the
catalytic cysteine on RING2 is blocked by
the UPD domain (Figure S1E) (46,54,55).
Phosphorylation of PARKIN (pPARKIN) and
pUb binding to PARKIN/pPARKIN disrupt
these autoinhibitory conformations and
with
autosomal-recessive
juvenile
Parkinsonism (AR-JP) and have been
extensively characterized in vitro and in
cells (1). In the case of PARKIN these
mutations are characterized by diverse
translocation and biochemical phenotypes
(1,23-33). It is currently thought that
pharmacological activators of PINK1 and/or
PARKIN may provide potential therapy to
treat Parkinson’s disease. For example it
2

activate PARKIN. Interestingly, while the
activation roles of PINK1 and pUb have
been investigated, the role of protein
substrates in PARKIN catalytic turnover
have escaped attention. PARKIN substrates
can provide acceptor lysines that clear
PARKIN~Ub thioester, thus facilitating
PARKIN enzyme turnover (56).
instrumentation beyond typical laboratory
operations.
To
begin
addressing
these
challenges, we previously demonstrated
that a ubiquitin C-terminal thioester probe
(ubiquitin
mercaptoethanesulfonate,
UbMES) and its fluorescent analogue
UbFluor could bypass the need for E1,
ATP, and E2 enzymes, thereby simplifying
assessment of HECT E3 ligase activity in
vitro (Figure 1) (58). Since the resulting
system bypasses the need for ATP, E1, and
E2 enzymes, we called it bypassing system
or ByS. We reasoned that the same system
could be useful for RBRs, as RBR ligases
also form an obligate E3~Ub thioester prior
to ligation. However, two unique features of
RBR E3 ligases require consideration. First,
unlike HECT ligases, RBRs are cysteine-
rich. For example, human PARKIN has 20
surface-exposed cysteines that could
Most importantly, there is a lack of
methods to a robust and simple quantifying
of PARKIN and other RBR E3s activity
which is needed for both biochemical
studies and small molecule screens. Typical
in vitro ubiquitination assays use a
reconstituted native cascade composed of
at least 5 components: E1, E2, E3,
ubiquitin, ATP, and a substrate, if the one is
used. In this setting, the assay is
operationally complex, expensive, and there
are many enzyme intermediates that makes
it difficult to conduct biochemical studies.
Coupled with the complex autoregulatory
mechanisms that govern the function of
PARKIN and other RBR E3s, the
complexity of native ubiquitination assays is
a major bottleneck in assessing the activity
of RBR E3 enzymes.
potentially
undergo
non-specific
transthiolation with Ub-MES (46). Secondly,
PARKIN and other RBR ligases have
complex, multistep activation mechanisms,
and it was not clear at the outset of this
work whether Ub-MES and its analogues
could recapitulate these mechanisms. For
example, it was not clear if UbMES that
lacks E2 enzyme, could sense activating
mutations that disrupt REP:RING1 interface
and open the E2 enzyme binding site.
Recently developed electrophilic
activity-based probes such as UbVME (55)
and electrophilic E2~Ub thioester mimics
(57) substantially reduce the complexity of
PARKIN and other catalytic cysteine
containing E3 ligase assays. However,
these probes are stoichiometric suicide
inhibitors, and therefore do not report on
catalytic turnover, lack high throughput
capabilities, and rely on western blotting for
quantitation. Absolute Quantification Mass
In this work, we report that E2-
independent ByS probes can be directly
and specifically charged to the catalytic
cysteine of PARKIN or pPARKIN, resulting
in native-like autoubiquitination, substrate
ubiquitination, and polyUb chain formation.
Spectrometry
quantification
(AQUA-MS)
of polyubiquitin
enables
chain
We further adapted the E2-
independent ByS to create UbFluor, a
quantitative probe for assessment of
PARKIN activity in vitro and for high-
throughput screening to identify PARKIN
activator compounds (Figure 1, Scheme 1).
UbFluor is a fluorescent thioester that
formation by PARKIN over multiple rounds
of Ub conjugation in vitro.(8) While this
method offers exquisite sensitivity, it is not
amenable to high-throughput screening,
and it requires considerable expertise and
3

features a fluorescein thiol that is attached
to the C-terminus of the ubiquitin via a
thioester bond. Effectively, UbFluor mimics
an E2~Ub thioester, except E2 enzyme is
replaced with fluorescein. When UbFluor is
charged to PARKIN’s catalytic cysteine, it
releases fluorescein thiol much like Ub~E2
releases E2 enzyme upon transthiolation.
The fluorescein release can be detected by
fluorescence polarization (FP). Thus,
fluorescence-based detection is built-in to
the transthiolation reaction, enabling real-
time monitoring of PARKIN catalytic
turnover, while eliminating any need for
extra reagent transfer steps or SDS-PAGE
analysis. UbFluor can be used to analyze
transthiolation under single turnover
reaction conditions (excess PARKIN),
similar to electrophilic Ub-VS/E2-Ub probes
or an E2~Ub discharge assay, or under
multiple turnover reaction conditions
(excess UbFluor) similar to the native
ubiquitination assays. Using UbFluor, we
verified PARKIN activation by Ser65
phosphorylation and by pUb binding,
demonstrating that the ByS is sensitive to
the native autoregulation mechanisms
governing PARKIN activity. We also
discovered that the PARKIN substrate
Miro1 and pUb cooperatively increase the
ubiquitin conjugation rate of pPARKIN.
Thus, while most work in the area has
focused on the roles of PINK1 and pUb on
parkin activation, our results suggest that
PARKIN substrates such as MIRO can also
enhance PARKIN catalytic turnover.
UbFluor as
a
probe to study the
biochemistry of PARKIN and other RBR E3
ligases and as a high-throughput screening
tool to identify small molecules that target
these enzymes.
Results
A
chemically
activated
ubiquitin
thioester recapitulates PARKIN function
in a simplified two-component reaction.
We first examined how well the E2-
independent ByS reaction recapitulates
PARKIN activity in the native cascade. To
test if the UbMES-based ByS recapitulates
Ser65 phosphorylation-dependent activation,
we prepared human PARKIN that was
phosphorylated by Tribolium castaneum
PINK1 (TcPINK1) on Ser65 (pPARKIN,
Figure S1A and B) (8) and ubiquitin
mercaptoethanesulfonate
(UbMES) (59) as previously described
(Figure S1C). Miro1 (Mitochondrial Rho 1)
thioester
is
a
calcium-sensitive regulator of
mitochondrial dynamics on the outer
mitochondrial membrane.(60) Miro1 is a
known substrate that is modified by
pPARKIN both in cells and in in vitro
assays.(5,14,61) We prepared full-length
human Miro1 (hMiro1) as well as an N-
terminally truncated construct (MiroS) that
is expressed more robustly and can be
purified in larger amounts than hMiro1
(MiroS; hMiro1 residues 177-592 with C-
terminal 6⋅His tag, 51.6 kDa, Figure
S1D).(62)
Lastly,
we
demonstrated
that
UbFluor can detect unnatural activation or
inactivation of PARKIN by point mutations
that disrupt either the REP:RING1 domain
or the UPD:RING2 domain interface, or
activation of PARKIN that lacks Ubl and
We
examined
MiroS
pPARKIN
substrate
autoubiquitination,
ubiquitination, and polyubiquitin chain
formation using UbMES (Figure 2). In the
presence
of
UbMES,
pPARKIN
UPD domains all together,
validating the biochemical relevance of the
UbFluor probe toward PARKIN
mechanisms. These results validate
further
ubiquitinated both itself and MiroS (Figure
2A lanes 4 and 6, respectively). In spite of
having ~28 surface lysines (Figure S1D),
4

MiroS was not modified by UbMES in the
absence of pPARKIN (Figure 2A, Lane 5).
To show that the observed ubiquitination
requires UbMES thioester functionality, we
We next examined ubiquitin chain
formation by pPARKIN under the ByS
reaction. Previous work demonstrated that
pPARKIN can form Lys48-, Lys63-, Lys6-,
and Lys11-linked chains in vitro.(8) We
compared PARKIN ubiquitination using
conducted
similar
experiments
with
hydrolysed UbMES (ubiquitin) indicated as
Hyd. (Figure 2A). Importantly, both
pPARKIN autoubiquitination and MiroS
substrate ubiquitination by UbMES were
strictly dependent on the catalytic cysteine
(Cys431) of pPARKIN. Therefore, despite the
fact that PARKIN is extremely cysteine-rich,
UbMES is selective for the catalytic site
cysteine. (Figure 2A, Lane 7).(46,62)
UbMES,
Ub(K48R)MES,
and
Ub(K63R)MES in the ByS reaction. For all
of these Ub variants, similar amounts of
ubiquitin adducts are formed (Figure 2D).
Antibodies against either Lys48- or Lys63-
linkages detected the corresponding chains,
which were absent when Ub(K48R)-MES or
Ub(63R)-MES, respectively, were used.
MALDI analysis confirmed the presence of
both K63- and K48-linkages, similar to the
native cascade reaction (Figure 2E).
Importantly, this experiment directly shows
that the E2 enzyme is dispensable for the
formation of polyubiquitin chains by
PARKIN under the ByS, consistent with our
previous report. (63)
Next, we demonstrated that PARKIN
ubiquitination using the ByS is still subject
to its native phosphoregulation mechanism
(Figure 2B). While both autoubiquitination
and MiroS substrate ubiquitination by wild-
type PARKIN were strongly activated by
TcPINK1
mutation of Ser65 to alanine (PARKINS65A
abolished this activation. Both
phosphorylation
(pPARKIN),
)
We next compared the sites within
full-length hMiro1 that are ubiquitinated by
pPARKIN either under ByS or native
reaction conditions, using tandem mass
spectrometry analysis. We limited hMiro1
modification to ~2-3 ubiquitin molecules in
both the ByS and in the native cascade,
and found that Lys153, Lys330, and Lys572
within hMiro1 were modified in both
reactions (Figure S2). These lysines have
been previously reported as ubiquitination
sites within hMiro1.(5,6,14,64) Because the
ByS reaction recapitulates the activity of
pPARKIN towards target lysine residues
within hMiro1, we can conclude that
pPARKIN alone can select target substrate
lysines without the help of an E2 enzyme in
vitro. Testing a larger panel of different
substrates could further confirm this finding.
Taken together, our results suggest that the
E2-independent ByS reaction recapitulates
several aspects of pPARKIN’s native
function: ubiquitin ligation, polyubiquitin
chain formation, polyubiquitin chain type
nonphosphorylated wild-type PARKIN and
PARKINS65A treated with TcPINK1 showed
only
a
minimal amount of MiroS
ubiquitination with UbMES (Figure 2B,
lanes 1-2 and 5-6, respectively). Mutation of
the catalytic cysteine Cys431 to alanine also
abolished PARKIN activity even after
TcPINK1 phosphorylation (pPARKINC431A
,
Figure
2B,
lanes
7-8).
pPARKIN
ubiquitination of MiroS increased with
increasing concentrations of UbMES, but
without
pPARKIN,
MiroS-Ub
adduct
formation was negligible (Figure 2C). Only
after incubating for a 3-fold longer reaction
time (120 min) at 80-120 µM UbMES, a
faint band corresponding to the molecular
weight of MiroS-Ub adduct was observed
(Lane 12-14, Figure 2C). This result shows
that our standard reaction conditions (30
µM UbMES and 40 min reaction time at
room
temperature)
do
not
cause
nonspecific modification of PARKIN,
ubiquitin, or the MiroS substrate.
5

specificity, and selection of substrate
lysines.
prepared UbFluor, a probe that enables
quantitative analysis of PARKIN activity
using the ByS. UbFluor preparation is
described in the methods section and in
previously published procedures (Scheme
1, Figure S3) (65). When UbFluor is
charged to PARKIN’s catalytic cysteine, it
releases fluorescein thiol and this release
can be monitored in real time using FP
(Fluor-SH, Figure 3A).
Lastly, we showed that the reaction
conditions of the ByS can be adjusted to
approach the timescale of the native
ubiquitination reaction, such that similar
amounts of the reaction products are
formed on a similar timescale (Figure 2F).
For these experiments, we used the same
amount of ubiquitin species, PARKIN
enzyme, and MiroS substrate in each
reaction. The native cascade reaction
contained 100 nM UBE1, 1 µM UbcH7, 1
µM pPARKIN, 30 µM Ub, 4 mM ATP, and 1
µM MiroS. The ByS reaction contained 1
µM PARKIN, 30 µM UbMES, and 1 µM
MiroS. The similar timescales of MiroS
ubiquitination in the ByS using 30 µM
UbMES and in a native cascade reaction
with a maximum of 1 µM UbcH7~Ub
certainly do not indicate that UbMES is as
good a PARKIN substrate as UbcH7~Ub.
However, this result demonstrates that ByS
reactions proceed on a very reasonable
timescale, providing an experimentally
feasible and simplified ubiquitination
reaction. Furthermore, the developed ByS
system can be used to prepare site-specific
First, we tested whether UbFluor
releases free Fluor-SH in a pPARKIN-
dependent manner. When UbFluor (0.5 µM)
was mixed with beta-mercaptoethanol
(BME; Figure 3B, lane 2), cysteamine
(Figure S4, lane 2) or pPARKIN (5 µM,
Figure 3B, lane 4), the fluorescent band
corresponding to UbFluor (~9 kDa)
diminished in intensity while Fluor-SH
intensity increased, in a time-dependent
manner (Figure 3C, Figure S5). pPARKIN
harbouring a C431A mutation failed to
release fluorescein from UbFluor after 60
minutes, demonstrating that the release of
Fluor-SH from UbFluor requires the
catalytic cysteine of PARKIN (Figure 3B,
lane 6).
Next, we tested whether the
consumption of UbFluor can be measured
in real-time using FP (Figure 3D-E). As
expected, increasing the amount of
pPARKIN in the UbFluor reaction
accelerated the rate of FP decrease under
excess enzyme conditions (Figure 3D-E).
Early time point data (1 - 5 min) were fitted
to obtain initial rates of FP decrease at an
effectively constant concentration of
UbFluor (Figure S6). The initial rate of
UbFluor consumption increased linearly
with pPARKIN concentration, as expected
for a bimolecular reaction. Since UbFluor
lacks E2 enzyme we high Km values can be
expected, similar to HECT E3s for which
Km values were ~800 µM (65). These data
demonstrate that UbFluor provides a robust
mono-
or
diubiquitinated
PARKIN
substrates using PARKIN and UbMES
(Figure 2F lane 3). This could allow to
bypass challenges using regular protein
synthesis approaches. Slower timescale of
of the ByS system and its simplicity (i.e. just
two components E3 and UbMES) make it
easier to adjust the stoichiometry of
reagents.
UbFluor enables real-time monitoring of
PARKIN activity by FP.
Encouraged that UbMES specifically
recognized the PARKIN catalytic cysteine
and recapitulated several essential aspects
of PARKIN activity, we designed and
6

FP-based assay for real-time monitoring of
PARKIN activity.
We titrated 0-25 µM pUb into ST
(Figure 4A) and MT (Figure 4B) UbFluor
reactions using both PARKIN and
pPARKIN. Titrations are written as
PARKIN/pUb or pPARKIN/pUb, with the
titrated reaction component after the slash,
throughout this work. Without PARKIN or
pPARKIN present, the small amount of
background UbFluor consumption is not
affected by pUb concentrations up to 25 µM
(No Enz/pUb, Figure 4A and B). However,
addition of pUb increases the UbFluor
consumption rate of both PARKIN and
pPARKIN under both ST and MT conditions
(Figure 4A and B). The pUb-dependent
increase in UbFluor consumption by
PARKIN under ST conditions was small but
significant (~1.5 fold increase) (Figure S8A-
C). This slight activation of PARKIN by pUb
was not previously detected using Ub-
VS.(64) As expected based on previous
results, the pUb-dependent increase in
UbFluor consumption by pPARKIN was
much larger (~7 fold for ST conditions)
(pPARKIN/pUb, Figure 4A and B).(7,64)
Non-phosphorylated ubiquitin did not
increase UbFluor consumption by pPARKIN
UbFluor confirms the native activation
mechanism of PARKIN by Ser65-
phosphorylation and pUb.
Changing the ratio of UbFluor to E3
ligase
in
ByS
reactions
enables
examination of different aspects of the E3
ligase mechanism. In a single turnover
(ST) reaction with excess PARKIN (10 fold
exces of PARKIN over UbFLuor), the rate
of FP decay only reflects the rate of
transthiolation reaction between UbFluor
and PARKIN. However, under multiple
turnover (MT) conditions (10 fold excess of
UbFluor over PARKIN), the UbFluor
consumption rate (FP decay) should reflect
both transthiolation and isopeptide ligation
rates. Under normal MT conditions one
molecule of PARKIN can consume >1
molecule of UbFluor. However, if there is a
defect in the isopeptide ligation step,
PARKIN can only consume one molecule of
UbFluor, because the resulting inactive
PARKIN~Ub thioester will not be able to
react with the second molecule of UbFluor.
(pPARKIN/Ub,
Figure
4A
and
B),
emphasizing the importance of Ser65
phosphorylation of ubiquitin for activating
PARKIN. Together, these experiments
confirm that the UbFluor assay can detect
the natural Ser65 phosphorylation and pUb-
dependent activation mechanisms of
PARKIN.
We used both ST and MT reaction
conditions to examine PARKIN activation by
Ser65 phosphorylation and by pUb to test
whether the UbFluor assay can detect
these
known
PARKIN
activation
mechanisms, similar to the native reaction.
We prepared pUb according to previously
established methods (Figure S7).(64) ST
experiments were performed at 5 µM
PARKIN and 0.5 µM UbFluor, while MT
experiments were performed at 2 µM
PARKIN and 20 µM UbFluor. To measure
initial velocities during which <20% of
available UbFluor was consumed, ST
reactions were carried out for 5 minutes,
and MT reactions were carried out for 15
minutes.
pUb and substrate enhance pPARKIN
catalytic turnover rate by different
mechanisms.
Next, we examined how the inclusion
of substrate affects the catalytic turnover
rate of pPARKIN in the presence of a
saturating
concentration
of
pUb
(pPARKIN+pUb; 25 µM pUb in our assays),
which is the maximally activated state of
7

PARKIN in the native reaction that has
been previously described.(7) We titrated
increasing concentrations of MiroS into
pPARKIN+ pUb. While 15 µM MiroS did not
increase the ST rate of pPARKIN+pUb, it
did increase the MT rate 2.6 fold (Figure 4
C and D, 4G). In contrast to MiroS addition,
adding an additional 15 µM pUb to a total
concentration of 40 µM did not further
increase either the ST or the MT rate of
UbFluor turnover by pPARKIN (Figure S9).
The simplest interpretation of these results
is that the presence of the MiroS substrate
does not significantly increase the
transthiolation rate but does enhance the
ligation rate of pPARKIN+pUb. To
determine whether the MiroS-dependent
increase in pPARKIN+pUb activity under
MT conditions that we observed could be
due to lysine-dependent clearance of the
Ub thioester, we titrated free lysine into the
UbFluor reaction with pPARKIN+pUb in
place of MiroS. We note that UbFluor is
stable at 100 mM free lysine (Figure 4E and
F and Figure S10). Similar to MiroS,
addition of free lysine increased the MT rate
of pPARKIN+pUb (~2 fold), but not the ST
rate (Figure 4E and F). Based on these
results it is reasonable to conclude that the
MiroS-dependent increase in the MT rate
may be due to the increased ligation rate
permanent fluorophore at its N-terminus.
This fluorophore is for gel imaging purposes
only, and is not transferred or released from
FUb at any point. FUb can be activated by
E1, and then be transferred onto E2 and
then E3 enzyme. We evaluated the effects
of pUb on both E2-discharge (ST) and total
ubiquitination (MT) activity of pPARKIN
(Figure S11). In an E2-discharge assay, the
E2~FUb thioester adduct (UbcH7~FUb in
our assays) is separately prepared and
chased by excess pPARKIN with the
indicated amount of pUb, MiroS, or both
(Figure S11A). The appearance of a
fluorescent
band
at
~55
kDa
(pPARKIN~FUb and Miro-FUb, Figure
S11B) and the reduction in the 25 kDa
UbcH7~Ub
band
(Figure
S11C),
corresponded to transthiolation activity. The
total ubiquitination activity of pPARKIN
under native cascade conditions was
measured after adding pUb, MiroS, or both
(Figure S11D). The appearance of
fluorescent bands above 10 kDa (Figure
S11E) and the reduction in the FUb band at
10 kDa (Figure S11F) were quantified to
reveal total Ub turnover. We confirmed that
pUb enhances the transthiolation of
pPARKIN at least ~4 fold, while MiroS has
no effect under these end point assay
conditions (Figure S11A, lane 3 and 4). In
contrast, we observed that the total
ubiquitin consumption for pPARKIN+pUb
was ~2 fold lower than pPARKIN+MiroS
(Figure S11D, lane 3 and 4). These results
indicate that pUb is a strong allosteric
activator of pPARKIN transthiolation, but is
not a good ubiquitin acceptor compared to
MiroS.
that
facilitates
the
clearance
of
pPARKIN~Ub thioester, regenerating the
free enzyme able to consume another
equivalent of UbFluor. Furthermore, the
increase in UbFluor consumption of
pPARKIN+pUb upon lysine or MiroS
titration suggests that pUb may be a poor
substrate for the isopeptide ligation step
compared to MiroS or lysine.
We were unable to conclusively
determine whether or not MiroS addition
further enhanced the pPARKIN+pUb
transthiolation rate in the native reaction,
because transthiolation reactions using
pPARKIN+pUb were too fast to quantify in
the discharge assay (Figure S11A, lanes 3
We further confirmed that pUb
activates transthiolation of pPARKIN but is
a poor substrate for ligation using the native
reaction with fluorescent ubiquitin (FUb).
Fluorescent ubiquitin (FUb) is different from
UbFluor, as it features a ubiquitin that has a
8

and 5). Similar to the UbFluor MT reaction,
pPARKIN shows the highest ubiquitin
conjugation efficiency when both pUb and
MiroS are present in a native cascade
assay (Figure S11D, lane 5).
reaction. This difference may arise because
UbFluor detects catalytic site activation
apart from E2 binding, unlike the native
reaction. This catalytic site activation may
reflect (1) physical opening of the catalytic
site, exposing the catalytic cysteine (Cys431)
on RING2 that is otherwise occluded by the
UPD, (2) chemical activation of Cys431 by
neighbouring residues for transthiolation or
ligation reactions, or both. In contrast,
binding of the E2~Ub complex is strictly
required to observe any activation in the
Together, all of these data suggest
that pUb activates pPARKIN by increasing
its transthiolation rate in both the E2-
independent UbFluor reaction and in the
native cascade. However, substrate is
required
for
maximal
pPARKIN
ubiquitination turnover even in the presence
of pUb, because pUb is a poor Ub acceptor
for pPARKIN. These results also confirm
that UbFluor ST and MT assays can
conveniently reveal different aspects of
PARKIN ubiquitination mechanisms: ST
assays detect transthiolation while MT
assays detect both transthiolation and
ligation. Furthermore, our results reveal a
similar trend between the UbFluor assay
and the native reaction under both ST and
MT conditions.
native reaction.
Therefore, the larger
difference between minimal and maximal
activity in the native reaction may be due to
multiple regulation sites: the E2~Ub
interaction site and the catalytic site (Figure
S1E).
Aside from higher basal PARKIN
activity in the UbFluor assay, the agreement
between these two measurements is
remarkable. Both measurements find that
activation of PARKIN by pUb is less than
activation by Ser65 phosphorylation, while
the greatest activation occurs with both pUb
and pPARKIN. This set of findings poses an
interesting question: how can UbFluor
reproduce the activity trend of PARKIN
observed in the native reaction, despite
using a distinct transthiolation mechanism?
It may be that while E2 binding clearly does
not happen in the ByS, the E2-independent
UbFluor transthiolation reaction is still
relevant to the mechanism of Ub binding
during the native transthiolation.
Comparison of UbFluor quantitation of
PARKIN
activity
with
previous
measurements using the native reaction.
Figure 4G compares the maximal
rates of UbFluor consumption that we
observed in this work to previous AQUA-MS
based measurements, which have provided
a quantitative measure of PARKIN activity
using
the
native
reaction.(7,64)
pPARKIN+pUb gave the highest PARKIN
activity in these experiments, therefore, we
set the activity of pPARKIN+pUb as a
standard value (100%) for comparison.
Consistent with this conclusion,
Kumar et al. showed that UbcH7-Ub binds
to pPARKIN 20-fold tighter than UbcH7
alone, suggesting that ubiquitin binding
itself has an important role in native
transthiolation.(66) Furthermore, recent
work showed that RING2 and RING1
domains of RBR E3s harbors ubiquitin
binding site (48). RING2 ubiquitin binding
site is particularly interesting since it was
The apparent activity of non-
phosphorylated PARKIN is much higher in
the UbFluor assay than in the native
reaction experiments. PARKIN activity is
~10% of pPARKIN+pUb activity in the
UbFluor assay, while it is only 0.02% of
pPARKIN+pUb activity in the native
9

shown that it recruits E2~Ub thioester for
transthiolation reaction. In this respect
RING2 domain is functionally similar to the
C-lobe of HECT E3 ligases that also has a
Ub binding site near the catalytic cysteine.
The latter serves to recruit E2~Ub thioester
PARKINS65E and pPARKIN using both the
UbMES-based ByS and the native reaction
(Figure 5 C and D). In both reactions,
pPARKIN robustly ubiquitinated MiroS while
PARKINS65E had only slight activity (Figure
5 C and D, Lane 2 and 8). As we previously
for
transthiolation
reaction
and
described
for
the
UbFluor
assay,
subsequently retains Ub in HECT E3~Ub
thioester for isopeptide ligation step (67-69).
unphosphorylated PARKIN showed higher
background activity in the UbMES-based
ByS reaction than in the native reaction
(Figure 5C, lane 6). The PARKINS65E
mutation
did
not
increase
Miro
S65E is a poor mimetic of pPARKIN.
ubiquitination significantly above this
background in the ByS. This result supports
our conclusion that S65E is not a good
mimetic of pPARKIN. While this work was in
progress, Ordureau et.al. published similar
findings (7).
Several
studies
have
used
overexpressed PARKINS65E in cells as a
phosphomimetic for pPARKIN, but it is
unclear whether PARKINS65E is activated
similar to pPARKIN. PARKINS65E failed to
show increased free ubiquitin chain
formation compared to PARKIN in a
Western blot assay.(7) In apparent contrast,
Kumar et al. demonstrated enhanced
UbFluor can detect PARKIN activation
by point mutations.
polyubiquitin
chain
formation
by
PARKINS65E compared to PARKIN.(66) We
PARKIN is autoregulated both by
mechanisms that block E2~Ub binding and
by mechanisms that occlude the catalytic
site. Because the UbFluor-based ByS
reaction does not include an E2, we may
expect it to only detect activation of the
catalytic site (Figure S1E). In fact, when
used with truncated HECT E3s, the ByS
detects mutations that alter activity near the
Ub binding site on the C-lobe and near the
catalytic cysteine, but not those that alter
E2-binding (65). However, for the RBR
ligase PARKIN, one report has postulated
that the REP:RING1 interface and the
RING2:UPD interface are allosterically
coupled (11,20). For example it was shown
that disrupting REP:RING1 interface with
W403A mutation mimics phosphorylated
PARKIN. This result suggests that changes
in regulation of the E2~Ub binding site may
translate to changes in the catalytic site,
such that these changes may be
observable using the ByS.
used
the
UbFluor-based
examine whether
ByS
to
pUb
quantitatively
activates PARKINS65E as it does for
pPARKIN.
We
performed
titration
experiments using PARKINS65E under the
same ST and MT conditions as defined for
pPARKIN in Figure 4 (Figure 5A and B).
pUb slightly increases the UbFluor
consumption rate of PARKINS65E under both
ST and MT conditions. This slight, < 1.5-
fold change under ST conditions is similar
to what we observed for pUb activation of
PARKIN under ST conditions (Figure S8A),
but is significantly smaller than the ~7- and
3-fold activation of pPARKIN by pUb under
ST and MT conditions, respectively (Figure
5A and B). These results indicate that S65E
does not mimic Ser65 phosphorylation of
pPARKIN in terms of activation by pUb
binding.
We further compared the MiroS
substrate
ubiquitination
efficiency
of
10

To determine the extent to which
UbFluor can detect different mechanisms of
PARKIN activation, we prepared five
constructs: four harbouring point mutations
that are known to activate PARKIN, and
one harbouring a mutation that abolishes
ubiquitination by fully active PARKIN. We
assume that the activating mutations
represent potential modes of activation by
small molecules. Activating mutations
disrupt (1) the REP:RING1 interface (A398T
and W403A)(46,54), or (2) the UPD:RING2
interface (F463Y and ΔUPD; the ΔUPD
construct consists of PARKIN residues 219-
465).(46) The inactivating mutation C431A
ablates the catalytic cysteine residue(43)
(Figure S1E). Importantly A398T is a
Parkinson’s disease mutation (1). To enable
a direct comparison with past data
evaluating the effects of these mutations on
PARKIN activity, PARKIN point mutations
were prepared using a base construct of rat
PARKIN 141-465 with an N-terminal GST
tag (ΔUbl, Figure S1E). The ΔUbl construct
lacks the ubiquitin-like domain and linker (1-
140), and therefore represents a minimally
autoregulated construct that still harbours a
low level of ubiquitination activity.(46)
activation by the REP:RING1 disrupting
mutation A398T and more robust activation
by W403A (compare Figure 6A lanes 10-12
and 13-15 to lanes 1-3). While there is no
E2~Ub bound structure of PARKIN, a
simple explanation for these results is that
the
autoinhibition of the Ub binding site in
PARKIN which is occupied by
W403A
mutation
may
relieve
UbMES/UbFluor to a larger extent than
A398T, or to expose the catalytic cysteine
of RING2 domain to a larger extent.
Regardless of the unknown structural
mechanisms by which these mutations
activate PARKIN, our W403A result
demonstrates that the E2-independent ByS
can detect PARKIN catalytic site activation
by significant disruption of the REP:RING1
interface, where E2 enzyme presumably
binds, and which is ~50 Å away from the
catalytic cysteine of PARKIN.
We next investigated whether the
same unnatural activation mechanisms
could be detected using the UbFluor assay.
To generate a quantitative comparison
between mutants, we determined the
apparent bimolecular rate of UbFluor
consumption (kobs) for each mutant. First,
we performed UbFluor assays using four
different concentrations of UbFluor under
either ST conditions (1 μM rat PARKIN with
0.25, 0.5, 0.75 and 1 μM UbFluor) or MT
conditions (5 μM rat PARKIN with 10, 12.5,
15 and 20 μM UbFluor). Each condition was
repeated twice for a total of eight
measurements. Next, we obtained the initial
UbFluor consumption rate by performing a
linear fit of the raw FP decrease at early
timepoints (1-5 min for ST; 1-15 min for MT;
Figure S12A). We then performed a linear
fit of the initial UbFluor consumption rate vs.
the UbFluor concentration (Figure S12B).
Finally, the slope of this linear fit was
divided by the PARKIN concentration to
obtain kobs (Figure 6B for MT and Figure
S13 for ST).
We first confirmed that the E2-
independent ByS recapitulates the known
properties of Ubl, ΔUPD, and ΔUPD
(C431A) using UbMES (Figure 6A). As
previously reported from native assays,(46)
Ubl exhibited a small amount of activity,
UPD had robust activity, and the C431A
mutation completely abolished the activity
of ΔUPD, demonstrating that the activity of
UPD in the ByS assay requires the catalytic
cysteine (Lane 1-3, 4-6 and 7-9
respectively, Figure 6A). Next, we
examined three activating point mutations in
the ΔUbl background using UbMES. As
expected, the F463Y mutation that disrupts
the UPD:RING2 interaction showed obvious
activation (compare Figure 6A lanes 16-18
to lanes 1-3). We observed modest
11

Using this bimolecular rate analysis,
we found that UbFluor detected activation
of the ΔUPD construct, as well as activation
of the ΔUbl construct by F463Y and W403A
mutations under both ST and MT
conditions, similar to the UbMES-based
ByS (Figure 6B and Figure S13,
respectively). The weak activation by
A398T observed in the UbMES assay was
not significant enough to be detected in the
UbFluor assay, as the activation was
smaller than the error in the kobs
consumption by pPARKIN and GST-tagged
HHARI RBR E3 ligases in a 384 well plate
using a microplate reader (Synergy 4,
Figure S16). Each E3 ligase (1.0 μM) was
incubated with either DMSO (0.2%;
negative control) or iodoacetamide (1 mM;
positive control) for 1 hour. UbFluor (5 μM)
was then added and endpoint FP readings
were recorded after 1 and 2 hours of
reaction. The 2-3-fold difference between
the endpoint values of these positive and
negative controls further demonstrates the
feasibility of using UbFluor for high-
throughput screening of bioactive molecules
for RBR E3 ligases under MT conditions.
measurement.
We applied the same
method to compare kobs of S65E and
pPARKIN, and found that in contrast to
phosphorylation, the S65E mutation did not
significantly increase PARKIN catalytic
turnover under either ST or MT conditions
(Figure S14).
Discussion
We began this work by applying our
previously developed UbMES-based ByS
assay to study the RBR E3 ligase PARKIN
(65). Despite the fact that PARKIN, like
other RBRs, is very cysteine-rich (45), we
found that the reaction with UbMES was
highly specific for the catalytic cysteine and
that the E2-independent bypassing system
recapitulates many aspects biochemical
mechanisms of PARKIN. Our results
demonstrate that in the absence of any E2
enzyme, pPARKIN can build polyUb chains
with specific linkages and select target
lysine residues within substrates in vitro.
Together, these results demonstrate
that UbFluor can recapitulate the majority of
the effects of PARKIN mutations observed
using UbMES. In addition, these results
confirmed that UbFluor assays are robust to
changes in the UbFluor concentration. We
determined kobs values using initial rates
obtained from four different UbFluor
concentrations under both ST and MT
conditions, and found that the rate of each
mutant relative to the wild type ΔUbl
construct is conserved at all UbFluor
concentrations used (Figure S15).(70) The
robustness of the UbFluor assay to different
concentrations of probe validates its use for
single-concentration and end-point high-
throughput screening assays.
After confirming that the E2-
independent ByS reaction recapitulated the
native activity of PARKIN, we used the
novel probe UbFluor to quantitatively
evaluate PARKIN activation by both natural
mechanisms (pUb and substrate) and
unnatural mechanisms (PARKIN mutations
that disrupt REP:RING1 and UPD:RING2
interface). Simple titration experiments
using UbFluor enabled us to quantitatively
compare and rank the native activation
mechanisms acting on PARKIN: Ser65-
phosphorylation and pUb. Our titration
Finally, after determining that the
UbFluor-based ByS can quantitatively
detect both natural and unnatural activation
mechanisms of the RBR E3 ligase PARKIN,
we performed an initial test of the dynamic
range of the UbFluor assay for high-
throughput screening to identify small
molecules that affect RBR E3 ligase
activity.
We
monitored
UbFluor
12

experiments also provided new insight on
pPARKIN+pUb activity. We found that
probe for high-throughput screening, as our
goal is to identify molecules that increase
ligation efficiency as well as the
transthiolation step.
although
pUb
strongly
activates
transthiolation step of PARKIN and
E2~Ub/UbFluor, maximal ubiquitination
turnover by pPARKIN is only observed
when a substrate or free lysine is present,
suggesting that pUb itself is not a good
Lastly, we demonstrated several
properties of the UbFluor assay that
indicate its suitability for high-throughput
screening to identify small molecules
affecting RBR E3 ligase activity. We found
that the E2-independent UbFluor reaction
can, at least in some cases, detect
significant activation at the REP:RING1 site
of PARKIN. In addition, the UbFluor-based
end-point assay detected activity changes
of both PARKIN and HHARI caused by
iodoacetamide with a strong signal change
in a 384 well-plate format. Because UbFluor
is stable at 100 mM lysine, direct
consumption of UbFluor by small molecules
containing amines is not expected.
However, because high concentrations of
free thiols (such as BME and cysteamine)
release Fluor-SH, small molecules with free
thiols should be avoided. Overall, these
ubiquitin
acceptor
for
pPARKIN.
Importantly, most efforts on studying the
mechanisms of PARKIN activation have
been focused on the activating role of
PINK1 and pUb. Here we show that
substrates that provide acceptor lysines can
also enhance PARKIN catalytic turnover.
Thus the local concentration of the
substrate can affect Parkin activity as
evident from ByS and native ubiquitination
systems (Figure 4G, S11).
We would like to highlight the
remarkable simplicity of the assay: only two
reagents are needed, and by changing the
ratio of UbFluor and enzyme we can
quantify transthiolation and isopeptide
ligation steps. Furthermore, we can easily
test the effect of other protein cofactors
such as pUb and substrates such as Miro
on PARKIN activity, and investigate their
roles on the enzyme mechanism (i.e.
activation of transthiolation step vs
isopeptide ligation step). We envision that
our findings will expand the use of UbFluor
probe for a robust quantifying of the
enzymatic activity of HECT, RBR, and
bacterial HECT-like and NEL E3 ligases,
upon structural mutations or in the presence
of protein partners such as pUb or
substrates.
results provide
demonstration that UbFluor can be used in
highly simplified high-throughput
a
very encouraging
a
screening assay to detect allosteric
activators or inhibitors of PARKIN and
perhaps other RBR E3 ligases. Further use
of
UbFluor
in
application
to
HECT/RBR/HECT-like bacterial/NEL E3s
will be reported in the future.
Acknowledgements
Funding from Northwestern University and
CLP Cornew Innovation Award is gratefully
acknowledged. A.V.S. is a Pew Scholar in
the Biomedical Sciences, supported by the
Pew Charitable Trusts. D.T.K. has been
supported by National Institute of General
Importantly, our data demonstrated
that UbFluor can detect changes in the
ligation efficiency of PARKIN under MT
conditions, despite the fact that the signal
Medical
Sciences
Training
Grant
5T32GM008382. D.T.K. is supported by an
NU Nicholson fellowship. A.V.S, S.E.R, and
S.J.P. are supported by a Michael J. Fox
Foundation Target Validation Pilot Award.
change
from
FP
occurs
during
transthiolation (Figure 4 and Figure S11).
This is an essential property of the UbFluor
13

Research reported in this publication was
supported by NIGMS of the National
Institutes of Health under award number
R01GM072656
(S.E.R.)
and
R01GM115632 (A.V.S.). The content is
solely the responsibility of the authors and
does not necessarily represent the official
views of the National Institutes of Health.
The authors declare that they have no
conflicts of interest with the contents of this
article.
Author Contributions
S.P., S.E.R, S.A.V. designed the study.
S.P., P.F., and D.T.K.
conducted
experiments, and collected the data. S.P.,
P.F., D.T.K. S.E.R., and S.A.V. analyzed
the data. The manuscript was written by
S.E.R., S.P., and S.A.V. All authors
participated in discussions about the
manuscript and approved the final version.
To whom correspondence should be
addressed: Tel. 713-743-9616; E-mail:
avstatsy@central.uh.edu
14

References
1.
2.
Pickrell, A. M., and Youle, R. J. (2015) The Roles of PINK1, Parkin, and Mitochondrial
Fidelity in Parkinson's Disease. Neuron 85, 257-273
Jin, S. M., Lazarou, M., Wang, C. X., Kane, L. A., Narendra, D. P., and Youle, R. J.
(2010) Mitochondrial membrane potential regulates PINK1 import and proteolytic
destabilization by PARL. Journal of Cell Biology 191, 933-942
3.
4.
5.
6.
7.
Meissner, C., Lorenz, H., Weihofen, A., Selkoe, D. J., and Lemberg, M. K. (2011) The
mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1
trafficking. J Neurochem 117, 856-867
Narendra, D. P., Jin, S. M., Tanaka, A., Suen, D. F., Gautier, C. A., Shen, J., Cookson,
M. R., and Youle, R. J. (2010) PINK1 Is Selectively Stabilized on Impaired Mitochondria
to Activate Parkin. Plos Biol 8
Sarraf, S. A., Raman, M., Guarani-Pereira, V., Sowa, M. E., Huttlin, E. L., Gygi, S. P.,
and Harper, J. W. (2013) Landscape of the PARKIN-dependent ubiquitylome in
response to mitochondrial depolarization. Nature 496, 372-+
Bingol, B., Tea, J. S., Phu, L., Reichelt, M., Bakalarski, C. E., Song, Q., Foreman, O.,
Kirkpatrick, D. S., and Sheng, M. (2014) The mitochondrial deubiquitinase USP30
opposes parkin-mediated mitophagy. Nature 510, 370-375
Ordureau, A., Heo, J. M., Duda, D. M., Paulo, J. A., Olszewski, J. L., Yanishevski, D.,
Rinehart, J., Schulman, B. A., and Harper, J. W. (2015) Defining roles of PARKIN and
ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin
replacement strategy. Proc Natl Acad Sci U S A 112, 6637-6642
15

8.
Ordureau, A., Sarraf, S. A., Duda, D. M., Heo, J. M., Jedrychowski, M. P., Sviderskiy, V.
O., Olszewski, J. L., Koerber, J. T., Xie, T., Beausoleil, S. A., Wells, J. A., Gygi, S. P.,
Schulman, B. A., and Harper, J. W. (2014) Quantitative Proteomics Reveal a
Feedforward Mechanism for Mitochondrial PARKIN Translocation and Ubiquitin Chain
Synthesis. Mol Cell 56, 360-375
9.
Heo, J. M., Ordureau, A., Paulo, J. A., Rinehart, J., and Harper, J. W. (2015) The
PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of
OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy. Mol Cell 60, 7-
20
10.
11.
Yamano, K., Queliconi, B. B., Koyano, F., Saeki, Y., Hirokawa, T., Tanaka, K., and
Matsuda, N. (2015) Site-specific Interaction Mapping of Phosphorylated Ubiquitin to
Uncover Parkin Activation. Journal of Biological Chemistry 290, 25199-25211
Caulfield, T. R., Fiesel, F. C., Moussaud-Lamodiere, E. L., Dourado, D. F., Flores, S. C.,
and Springer, W. (2014) Phosphorylation by PINK1 Releases the UBL Domain and
Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin. PLoS Comput
Biol 10, e1003935
12.
13.
Shiba-Fukushima, K., Imai, Y., Yoshida, S., Ishihama, Y., Kanao, T., Sato, S., and
Hattori, N. (2012) PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain
primes mitochondrial translocation of Parkin and regulates mitophagy. Sci Rep-Uk 2
Wauer, T., Swatek, K. N., Wagstaff, J. L., Gladkova, C., Pruneda, J. N., Michel, M. A.,
Gersch, M., Johnson, C. M., Freund, S. M. V., and Komander, D. (2015) Ubiquitin Ser65
phosphorylation affects ubiquitin structure, chain assembly and hydrolysis. Embo
Journal 34, 307-325
16

14.
15.
Kazlauskaite, A., Kelly, V., Johnson, C., Baillie, C., Hastie, C. J., Peggie, M., Macartney,
T., Woodroof, H. I., Alessi, D. R., Pedrioli, P. G., and Muqit, M. M. (2014)
Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1
substrate-based assay of Parkin E3 ligase activity. Open Biol 4, 130213
Kondapalli, C., Kazlauskaite, A., Zhang, N., Woodroof, H. I., Campbell, D. G., Gourlay,
R., Burchell, L., Walden, H., Macartney, T. J., Deak, M., Knebel, A., Alessi, D. R., and
Muqit, M. M. (2012) PINK1 is activated by mitochondrial membrane potential
depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65.
Open Biol 2, 120080
16.
17.
Zheng, X. D., and Hunter, T. (2013) Parkin mitochondrial translocation is achieved
through a novel catalytic activity coupled mechanism. Cell Res 23, 886-897
Iguchi, M., Kujuro, Y., Okatsu, K., Koyano, F., Kosako, H., Kimura, M., Suzuki, N.,
Uchiyama, S., Tanaka, K., and Matsuda, N. (2013) Parkin-catalyzed Ubiquitin-Ester
Transfer Is Triggered by PINK1-dependent Phosphorylation. Journal of Biological
Chemistry 288
18.
19.
Koyano, F., Okatsu, K., Kosako, H., Tamura, Y., Go, E., Kimura, M., Kimura, Y.,
Tsuchiya, H., Yoshihara, H., Hirokawa, T., Endo, T., Fon, E. A., Trempe, J. F., Saeki,
Y., Tanaka, K., and Matsuda, N. (2014) Ubiquitin is phosphorylated by PINK1 to
activate parkin. Nature 510, 162-+
Kane, L. A., Lazarou, M., Fogel, A. I., Li, Y., Yamano, K., Sarraf, S. A., Banerjee, S.,
and Youle, R. J. (2014) PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin
ligase activity. Journal of Cell Biology 205, 143-153
17

20.
Aguirre, J. D., Dunkerley, K. M., Mercier, P., and Shaw, G. S. (2017) Structure of
phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation. P
Natl Acad Sci USA 114, 298-303
21.
22.
Wauer, T., Simicek, M., Schubert, A., and Komander, D. (2015) Mechanism of phospho-
ubiquitin-induced PARKIN activation (vol 524, pg 370, 2015). Nature 526, 728-728
Kazlauskaite, A., Kondapalli, C., Gourlay, R., Campbell, D. G., Ritorto, M. S., Hofmann,
K., Alessi, D. R., Knebel, A., Trost, M., and Muqit, M. M. K. (2014) Parkin is activated by
PINK1-dependent phosphorylation of ubiquitin at Ser(65). Biochem J 460, 127-139
Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S.,
Yokochi, M., Mizuno, Y., and Shimizu, N. (1998) Mutations in the parkin gene cause
autosomal recessive juvenile parkinsonism. Nature 392, 605-608
23.
24.
Lucking, C. B., Durr, A., Bonifati, V., Vaughan, J., De Michele, G., Gasser, T., Harhangi,
B. S., Meco, G., Denefle, P., Wood, N. W., Agid, Y., Brice, A., French Parkinson's
Disease Genetics Study, G., and European Consortium on Genetic Susceptibility in
Parkinson's, D. (2000) Association between early-onset Parkinson's disease and
mutations in the parkin gene. The New England journal of medicine 342, 1560-1567
Valente, E. M., Abou-Sleiman, P. M., Caputo, V., Muqit, M. M., Harvey, K., Gispert, S.,
Ali, Z., Del Turco, D., Bentivoglio, A. R., Healy, D. G., Albanese, A., Nussbaum, R.,
Gonzalez-Maldonado, R., Deller, T., Salvi, S., Cortelli, P., Gilks, W. P., Latchman, D. S.,
Harvey, R. J., Dallapiccola, B., Auburger, G., and Wood, N. W. (2004) Hereditary early-
onset Parkinson's disease caused by mutations in PINK1. Science 304, 1158-1160
Fiesel, F. C., Caulfield, T. R., Moussaud-Lamodiere, E. L., Ogaki, K., Dourado, D. F. A.
R., Flores, S. C., Ross, O. A., and Springer, W. (2015) Structural and Functional Impact
25.
26.
18

of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin. Hum
Mutat 36, 774-786
27.
28.
Safadi, S. S., and Shaw, G. S. (2007) A disease state mutation unfolds the parkin
ubiquitin-like domain. Biochemistry-Us 46, 14162-14169
Sriram, S. R., Li, X. J., Ko, H. S., Chung, K. K. K., Wong, E., Lim, K. L., Dawson, V. L.,
and Dawson, T. M. (2005) Familial-associated mutations differentially disrupt the
solubility, localization, binding and ubiquitination properties of parkin. Hum Mol Genet
14, 2571-2586
29.
30.
31.
32.
Hampe, C., Ardila-Osorio, H., Fournier, M., Brice, A., and Corti, O. (2006) Biochemical
analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein
ligase with monoubiquitylation capacity. Hum Mol Genet 15, 2059-2075
Henn, I. H., Gostner, J. M., Lackner, P., Tatzelt, J., and Winklhofer, K. F. (2005)
Pathogenic mutations inactivate parkin by distinct mechanisms. J Neurochem 92, 114-
122
Matsuda, N., Kitami, T., Suzuki, T., Mizuno, Y., Hattori, N., and Tanaka, K. (2006)
Diverse effects of pathogenic mutations of parkin that catalyze multiple
monoubiquitylation in vitro. Journal of Biological Chemistry 281, 3204-3209
Spratt, D. E., Martinez-Torres, R. J., Noh, Y. J., Mercier, P., Manczyk, N., Barber, K. R.,
Aguirre, J. D., Burchell, L., Purkiss, A., Walden, H., and Shaw, G. S. (2013) A molecular
explanation for the recessive nature of parkin-linked Parkinson's disease. Nature
Communications 4
33.
Kazlauskaite, A., Martinez-Torres, R. J., Wilkie, S., Kumar, A., Peltier, J., Gonzalez, A.,
Johnson, C., Zhang, J. W., Hope, A. G., Peggie, M., Trost, M., van Aalten, D. M. F.,
19

Alessi, D. R., Prescott, A. R., Knebel, A., Walden, H., and Muqit, M. M. K. (2015)
Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-
dependent phosphorylation and activation. Embo Rep 16, 939-954
34.
35.
36.
Clark, I. E., Dodson, M. W., Jiang, C., Cao, J. H., Huh, J. R., Seol, J. H., Yoo, S. J.,
Hay, B. A., and Guo, M. (2006) Drosophila pink1 is required for mitochondrial function
and interacts genetically with parkin. Nature 441, 1162-1166
Park, J., Lee, S. B., Lee, S., Kim, Y., Song, S., Kim, S., Bae, E., Kim, J., Shong, M.,
Kim, J. M., and Chung, J. (2006) Mitochondrial dysfunction in Drosophila PINK1
mutants is complemented by parkin. Nature 441, 1157-1161
Paterna, J. C., Leng, A., Weber, E., Feldon, J., and Bueler, H. (2007) DJ-1 and Parkin
modulate dopamine-dependent behavior and inhibit MPTP-induced nigral dopamine
neuron loss in mice. Molecular therapy : the journal of the American Society of Gene
Therapy 15, 698-704
37.
38.
39.
Bian, M., Liu, J., Hong, X., Yu, M., Huang, Y., Sheng, Z., Fei, J., and Huang, F. (2012)
Overexpression of parkin ameliorates dopaminergic neurodegeneration induced by 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. PloS one 7, e39953
Hertz, N. T., Berthet, A., Sos, M. L., Thorn, K. S., Burlingame, A. L., Nakamura, K., and
Shokat, K. M. (2013) A Neo-Substrate that Amplifies Catalytic Activity of Parkinson's-
Disease-Related Kinase PINK1. Cell 154, 737-747
Regnstrom, K., Yan, J. L., Nguyen, L., Callaway, K., Yang, Y. L., Diep, L., Xing, W. M.,
Adhikari, A., Beroza, P., Hom, R. K., Riley, B., Rudolph, D., Jobling, M. F., Baker, J.,
Johnston, J., Konradi, A., Bova, M. P., and Artis, R. D. (2013) Label Free Fragment
20

Screening Using Surface Plasmon Resonance as a Tool for Fragment Finding -
Analyzing Parkin, a Difficult CNS Target. Plos One 8
40.
41.
Wenzel, D. M., Lissounov, A., Brzovic, P. S., and Klevit, R. E. (2011) UBCH7 reactivity
profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105-U136
Lazarou, M., Narendra, D. P., Jin, S. M., Tekle, E., Banerjee, S., and Youle, R. J. (2013)
PINK1 drives Parkin self-association and HECT-like E3 activity upstream of
mitochondrial binding. The Journal of cell biology 200, 163-172
42.
43.
Zheng, X., and Hunter, T. (2013) Parkin mitochondrial translocation is achieved through
a novel catalytic activity coupled mechanism. Cell research 23, 886-897
Riley, B. E., Lougheed, J. C., Callaway, K., Velasquez, M., Brecht, E., Nguyen, L.,
Shaler, T., Walker, D., Yang, Y., Regnstrom, K., Diep, L., Zhang, Z., Chiou, S., Bova,
M., Artis, D. R., Yao, N., Baker, J., Yednock, T., and Johnston, J. A. (2013) Structure
and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases.
Nat Commun 4, 1982
44.
45.
Wauer, T., and Komander, D. (2013) Structure of the human Parkin ligase domain in an
autoinhibited state. The EMBO journal 32, 2099-2112
Trempe, J. F., Sauve, V., Grenier, K., Seirafi, M., Tang, M. Y., Menade, M., Al-Abdul-
Wahid, S., Krett, J., Wong, K., Kozlov, G., Nagar, B., Fon, E. A., and Gehring, K. (2013)
Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science 340,
1451-1455
46.
Trempe, J. F., Sauve, V., Grenier, K., Seirafi, M., Tang, M. Y., Menade, M., Al-Abdul-
Wahid, S., Krett, J., Wong, K., Kozlov, G., Nagar, B., Fon, E. A., and Gehring, K. (2013)
21

Structure of Parkin Reveals Mechanisms for Ubiquitin Ligase Activation. Science 340,
1451-1455
47.
Joselin, A. P., Hewitt, S. J., Callaghan, S. M., Kim, R. H., Chung, Y. H., Mak, T. W.,
Shen, J., Slack, R. S., and Park, D. S. (2012) ROS-dependent regulation of Parkin and
DJ-1 localization during oxidative stress in neurons. Human molecular genetics 21,
4888-4903
48.
49.
Dove, K. K., Stieglitz, B., Duncan, E. D., Rittinger, K., and Klevit, R. E. (2016) Molecular
insights into RBR E3 ligase ubiquitin transfer mechanisms. Embo Rep 17, 1221-1235
Duda, D. M., Olszewski, J. L., Schuermann, J. P., Kurinov, I., Miller, D. J., Nourse, A.,
Alpi, A. F., and Schulman, B. A. (2013) Structure of HHARI, a RING-IBR-RING Ubiquitin
Ligase: Autoinhibition of an Ariadne-Family E3 and Insights into Ligation Mechanism.
Structure 21, 1030-1041
50.
51.
52.
Stieglitz, B., Rana, R. R., Koliopoulos, M. G., Morris-Davies, A. C., Schaeffer, V.,
Christodoulou, E., Howell, S., Brown, N. R., Dikic, I., and Rittinger, K. (2013) Structural
basis for ligase-specific conjugation of linear ubiquitin chains by HOIP. Nature 503, 422-
+
Kelsall, I. R., Duda, D. M., Olszewski, J. L., Hofmann, K., Knebel, A., Langevin, F.,
Wood, N., Wightman, M., Schulman, B. A., and Alpi, A. F. (2013) TRIAD1 and HHARI
bind to and are activated by distinct neddylated Cullin-RING ligase complexes. Embo
Journal 32, 2848-2860
Lechtenberg, B. C., Rajput, A., Sanishvili, R., Dobaczewska, M. K., Ware, C. F., Mace,
P. D., and Riedl, S. J. (2016) Structure of a HOIP/E2 similar to ubiquitin complex
reveals RBR E3 ligase mechanism and regulation. Nature 529, 546-+
22

53.
Ho, S. R., Lee, Y. J., and Lin, W. C. (2015) Regulation of RNF144A E3 Ubiquitin Ligase
Activity by Self-association through Its Transmembrane Domain. Journal of Biological
Chemistry 290, 23026-23038
54.
55.
Wauer, T., and Komander, D. (2013) Structure of the human Parkin ligase domain in an
autoinhibited state. Embo Journal 32, 2099-2112
Riley, B. E., Lougheed, J. C., Callaway, K., Velasquez, M., Brecht, E., Nguyen, L.,
Shaler, T., Walker, D., Yang, Y., Regnstrom, K., Diep, L., Zhang, Z., Chiou, S., Bova,
M., Artis, D. R., Yao, N., Baker, J., Yednock, T., and Johnston, J. A. (2013) Structure
and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases.
Nature Communications 4
56.
57.
Bender, M. L., Kezdy, F. J., and Wedler, F. C. (1967) Alpha-Chymotrypsin - Enzyme
Concentration and Kinetics. J Chem Educ 44, 84-&
Pao, K. C., Stanley, M., Han, C., Lai, Y. C., Murphy, P., Balk, K., Wood, N. T., Corti, O.,
Corvol, J. C., Muqit, M. M., and Virdee, S. (2016) Probes of ubiquitin E3 ligases enable
systematic dissection of parkin activation. Nat Chem Biol
58.
59.
Park, S., Krist, D. T., and Statsyuk, A. V. (2015) Protein ubiquitination and formation of
polyubiquitin chains without ATP, E1 and E2 enzymes. Chemical Science 6, 1770-1779
El Oualid, F., Merkx, R., Ekkebus, R., Hameed, D. S., Smit, J. J., de Jong, A., Hilkmann,
H., Sixma, T. K., and Ovaa, H. (2010) Chemical synthesis of ubiquitin, ubiquitin-based
probes, and diubiquitin. Angew Chem Int Ed Engl 49, 10149-10153
Birsa, N., Norkett, R., Higgs, N., Lopez-Domenech, G., and Kittler, J. T. (2013)
Mitochondrial trafficking in neurons and the role of the Miro family of GTPase proteins.
Biochem Soc Trans 41, 1525-1531
60.
23

61.
62.
Wang, X., Winter, D., Ashrafi, G., Schlehe, J., Wong, Y. L., Selkoe, D., Rice, S., Steen,
J., LaVoie, M. J., and Schwarz, T. L. (2011) PINK1 and Parkin target Miro for
phosphorylation and degradation to arrest mitochondrial motility. Cell 147, 893-906
Klosowiak, J. L., Focia, P. J., Chakravarthy, S., Landahl, E. C., Freymann, D. M., and
Rice, S. E. (2013) Structural coupling of the EF hand and C-terminal GTPase domains
in the mitochondrial protein Miro. EMBO Rep 14, 968-974
63.
64.
Park, S., Boneh, G., and Statsyuk, A. V. (2014) Protein ubiquitination and formation of
polyubiquitin chains without ATP, E1, and E2 enzymes. Abstr Pap Am Chem S 248
Ordureau, A., Sarraf, S. A., Duda, D. M., Heo, J. M., Jedrychowski, M. P., Sviderskiy, V.
O., Olszewski, J. L., Koerber, J. T., Xie, T., Beausoleil, S. A., Wells, J. A., Gygi, S. P.,
Schulman, B. A., and Harper, J. W. (2014) Quantitative proteomics reveal a feedforward
mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol
Cell 56, 360-375
65.
66.
Krist, D. T., Park, S., Boneh, G. H., Rice, S. E., and Statsyuk, A. V. (2016) UbFluor: a
mechanism-based probe for HECT E3 ligases. Chemical Science 7, 5587-5595
Kumar, A., Aguirre, J. D., Condos, T. E., Martinez-Torres, R. J., Chaugule, V. K., Toth,
R., Sundaramoorthy, R., Mercier, P., Knebel, A., Spratt, D. E., Barber, K. R., Shaw, G.
S., and Walden, H. (2015) Disruption of the autoinhibited state primes the E3 ligase
parkin for activation and catalysis. EMBO J 34, 2506-2521
67.
Kamadurai, H. B., Souphron, J., Scott, D. C., Duda, D. M., Miller, D. J., Stringer, D.,
Piper, R. C., and Schulman, B. A. (2009) Insights into Ubiauitin Transfer Cascades from
a Structure of a UbcH5B similar to Ubiauitin-HECTNEDD4L Complex. Mol Cell 36,
1095-1102
24

68.
69.
Maspero, E., Valentini, E., Mari, S., Cecatiello, V., Soffientini, P., Pasqualato, S., and
Polo, S. (2013) Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis
for catalytic priming. Nature Structural & Molecular Biology 20, 696-+
Kamadurai, H. B., Qiu, Y., Deng, A., Harrison, J. S., MacDonald, C., Actis, M.,
Rodrigues, P., Miller, D. J., Souphron, J., Lewis, S. M., Kurinov, I., Fujii, N., Hammel,
M., Piper, R., Kuhlman, B., and Schulman, B. A. (2013) Mechanism of ubiquitin ligation
and lysine prioritization by a HECT E3. Elife 2
70.
71.
Eisenthal, R., Danson, M. J., and Hough, D. W. (2007) Catalytic efficiency and kcat/KM:
a useful comparator? Trends Biotechnol 25, 247-249
Chew, K. C., Matsuda, N., Saisho, K., Lim, G. G., Chai, C., Tan, H. M., Tanaka, K., and
Lim, K. L. (2011) Parkin mediates apparent E2-independent monoubiquitination in vitro
and contains an intrinsic activity that catalyzes polyubiquitination. PLoS One 6, e19720
Smit, J. J., Monteferrario, D., Noordermeer, S. M., van Dijk, W. J., van der Reijden, B.
A., and Sixma, T. K. (2012) The E3 ligase HOIP specifies linear ubiquitin chain
assembly through its RING-IBR-RING domain and the unique LDD extension. EMBO J
31, 3833-3844
72.
25

Figure/Scheme captions:
Figure 1. Chemical activation of the C-terminus of ubiquitin as a thioester (e.g. UbMES or
UbFluor) can bypass the need for E1, E2 and ATP, downsizing the 5-component native
cascade reaction (E1, E2, ATP, PARKIN, and Ub) to 2 components (PARKIN and ByS
probes). Transthiolation with UbMES releases a mercaptoethanesulfonate group (MES), while
transthiolation with UbFluor releases fluorescein thiol.
Scheme 1. Synthesis of UbFluor.
Figure 2. The PARKIN/ByS recapitulates several aspects of the native cascade. (A) ByS
reaction of pPARKIN, UbMES, and the MiroS substrate. Lane 4 shows pPARKIN
autoubiquitination and chain formation, while Lane 6 shows pPARKIN autoubiquitination, chain
formation, and MiroS substrate ubiquitination. Notably, UbMES has no ubiquitination activity in
the absence of pPARKIN, or in the presence of catalytically inactive C431A pPARKIN (lanes 5
and 7). Hyd. indicates hydrolyzed UbMES before the reaction. (B) pPARKIN undergoes
autoubiquitination and MiroS ubiquitination using UbMES. Mutation of the primary
phosphorylation site (S65A) or the catalytic cysteine (C431A) blocks this activity. P indicates
PARKIN, M indicates MiroS and * indicates impurities. PARKIN (S65A) was pretreated with
TcPINK1 under the same reaction conditions as PARKIN, prior to its use in assay. (C) ByS
ubiquitination depends on the concentration of UbMES and requires pPARKIN. (D)
pPARKIN/ByS forms K48- and K63-linked chains, similar to the native cascade. The reaction
with pPARKIN, MiroS and the corresponding UbMES were analyzed by western blot. A K48R
26

mutation in UbMES blocks formation of K48-, but not K63-linked chains (lane 3), while a K63R
mutation in UbMES blocks formation of K63-, but not K48-linked chains (lane 4). (E) MALDI-
TOF analysis of a slice from the stacking gel of a native cascade reaction and the ByS reaction
from (D), lane 2, confirming K48 and K63 chain formation. (F) MiroS ubiquitination by
pPARKIN using the ByS (1 µM pPARKIN and 30 µM UbMES) proceeds on a similar timescale
to common native reaction conditions (100 nM UBE1, 1 µM UbcH7, 1 µM pPARKIN, 30 µM
Ub, and 4 mM ATP). P indicates PARKIN and M indicates MiroS.
Figure 3. UbFluor reports pPARKIN activity in real-time by fluorescence polarization. (A)
Design of the ByS reaction using UbFluor and PARKIN. (B), BME (lane 2) or pPARKIN (lane 4)
can release free fluorescein (SFluor) from UbFluor but not pPARKINC431A (lane 6). (C) Gel-
based analysis of UbFluor/pPARKIN reaction. At the indicated time points, the reaction was
stopped by addition of non-reducing Laemmli buffer and analyzed by SDS-PAGE. A time-
dependent decrease in the intensity of the UbFluor band and increase in the free fluorescein
band were observed. (D) FP-based analysis of UbFluor (0.5 μM) consumption by the indicated
amount of pPARKIN (0, 1, 2, 3, 4, and 5 μM). Mean ± SEM of 3 measurements for each
concentration of pPARKIN are shown. (E) Plot of the initial rates (0-5 min) from (D) showing
that the rate of UbFluor consumption increases linearly with pPARKIN concentration
(R2=0.996).
27

Figure 4. UbFluor confirms multiple activation states of PARKIN. Titrations in the left panels
(A, C, and E) were performed under ST conditions (5 µM PARKIN, 0.5 µM UbFluor), while the
right panels (B, D, and F) were performed under MT conditions (2 µM PARKIN, 20 µM
UbFluor). Each titration was repeated twice and all data are shown. Solid lines join the rates
determined from one titration experiment, while dashed lines join rates determined from a
second titration. A-B, Ub or pUb titration into a UbFluor reaction with PARKIN or pPARKIN. C-
D, MiroS titration into pPARKIN+25 µM pUb. E-F, Lysine titration into pPARKIN+25 µM pUb.
G, Comparison of PARKIN activation measured using UbFluor with previous data. UbFluor
measurements under ST and MT conditions and SDS-PAGE based quantification are reported
in this work. AQUA-MS data were reported in by Ordureau et al(7,64). For comparison of
UbFluor to AQUA-MS, the maximal rate corresponding to each state is determined as a
percentage of pPARKIN+pUb activity. Cartoons depict the relative activity of each PARKIN
species, with unmodified PARKIN having the lowest activity and pPARKIN+pUb+MiroS having
the highest. * rate was too fast to accurately resolve.
28

Figure 5. S65E is a poor mimetic of pPARKIN. (A-B) ST (A) and MT (B) experiments titrating
Ub or pUb into a UbFluor reaction with PARKINS65E (green triangles) were compared to
PARKIN (orange circles) and pPARKIN (blue diamonds) titrations from Figure 4. Conditions
were as described in Figure 4. Each titration was repeated twice and all data are shown. Solid
lines join the rates determined from one titration experiment, while dashed lines join rates
determined from a second titration. (C-D) Ubiquitination of MiroS by pPARKIN, pPARKINC431A
PARKIN, or PARKINS65E were performed by (C) the UbMES-based ByS reaction or (D) the
native reaction. Conditions for these experiments were identical to those used in Figure 2F. *
indicates ubiquitinated MiroS.
,
Figure 6. UbFluor can quantify PARKIN activation or inactivation by known point mutations.
(A) UbMES assay on ΔUbl, ΔUPD, ΔUPD (C431A), and activating PARKIN mutations in the
ΔUbl background (A398T, W403A, and F463Y). Experiments with each mutant were
performed using 30 µM UbMES and 1.0 µM PARKIN for the time indicated. W403A and F463Y
were run on a separate gel. (B) Bar graph showing bimolecular reaction rates of UbFluor
consumption under MT conditions by PARKIN mutants as described above using the UbFluor
assay. Errors are based on linear fits of data from two repeats of UbFluor consumption data at
4 different concentrations of UbFluor (8 total measurements).
29

Figure 1
30