Purification of Rab5a Effector Responsible for Glutelin mRNA Location via
Affinity Chromatography
1
2
2
Alice La , Mio Sato , Thomas W. Okita .
1CalState
Fullerton; 2Institute of Biological Chemistry, Washington State Univ.
2010 REU in Interdisciplinary Plant Genomics
Rice is a major staple food source and as a source of dietary
protein to both human and livestock (1, 2). The major storage
proteins in rice (Oryza Sativa) are prolamine and glutelin, which are
synthesized on the endoplasmic reticulum (ER). In rice endosperm,
prolamin protein makes up 20-30% of total rice storage protein (1,
2). Prolamin mRNA is targeted to the PB-ER where it is translated
and then it forms intracisternal inclusions granules within the ER
lumen, a structure called PB-I (3, 4, 5). On the other hand, glutelin
makes up 60-80% of the protein content in endosperm (6, 2, 7).
Glutelin mRNAs are targeted to the cisternal ER. After they are
synthesized as 57kDa glutelin precursor (proglutelin), they are
transported to protein storage vacuoles (PSV) where they are
cleaved into 30-36kDa acidic and 19-22kDa basic subunits, thus
forming the PB-II (2, 3, 6, 8).
The glup4 mutant was induced by chemical mutagenesis using NMethyl-N-Nitrosourea (MNU) (7, 9). This mutant is characterized by
high proglutelin accumulation and the mis-localization of glutelin
mRNA to the PB-ER rather than to the cisternal ER (4, 7, 8). From
map-based cloning results, it was determined that this Glup4 gene
encodes a small GTPase Rab5a, a protein involved in membrane
vesicular transport (3, 7, 8). The Rab family functions as regulators of
distinct steps in membrane trafficking by recruiting specific effector
proteins onto membranes in their active GTP fixed form (10).
Through their effectors, Rab GTPases regulate vesicle formation,
actin- and tubulin-dependent vesicle movement, and membrane
fusion (10). Rab proteins are catalyzed by a GDP/GTP exchange
factor (GEF) (10). In the Drosophila egg, VPS22, 25, and 36 are part
of an ESCRT-II complex that is associated with vesicle formation and
bicoid mRNA localization (11). Therefore, rice Rab5a and VPS9 (GEF
for Rab5a) may have the ability to participate in the mRNA targeting
mechanism.
In order to learn more of the Rab5a associated trafficking process
involved, we set out to find Rab5a-binding proteins from rice seed
extract. In doing so, we expressed two Rab5a variants in E. coli. The
Rab5a-Q70L mutant is the constitutively active form and EM960, a
glup4 mutant line, contains a mutation (G45D) in its effector region,
thus preventing its role in specifying membrane vesicular transport
and targeting. We prepared affinity chromatography columns to
probe for the unknown effector protein which is essential for
membrane-vesicule associated RNA targeting (9). Results from SDSPAGE of elution fractions showed specific bands in the positive
control. However, given the low protein content, we have to
consider optimizing our experimental conditions.
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7
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Q70L EM960
72
72
55
55
43
43
34
34
Rab5a
Rab5a
26
26
GST
Tag
Fig. A1 Q70L Column
Fig. B1 Q70L column
Fig. A2 EM960 Column
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7
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7-2 8
Q70L EM960
72
55
43
34
26
Fig. A3 GST-tag column
Legend
Legend
1: T=0
2: T=3
3: ppt.
4: sup.
5: After filtration
6: FT
7: Wash (125 mL)
7-2: Wash (125mL excess)
8: Resin
1: Starting Material
2: FT
3: CSB buffer wash
(10mL)
4: Wash buffer A (40
mL)
5: Wash buffer B (50
mL)
6: Resin after Elute
GST Tag
Fig. B2 EM960 column
Column Preparation: The purified GST-Rab5a mutant proteins were incubated with
Glutathione Sepharose. To check for the binding efficiency of the mutant Rab5a to the
column, samples were collected at various points during the Rab5a mutant purification
and column preparation process as indicated in the legend. Fig. A1 is the positive Rab5aQ70L construct column. Fig. A2 is the negative construct column, EM960. Fig. A3 is the
GST-tag column used to crudely remove nonspecific binding in seed extract sample. The
50kDa band corresponds to GST-Rab5a mutant and the 26kDa band is the GST-tag.
Loading Columns with Rice Seed Extract: For all columns, lane 1
corresponds to the amount of starting material after simple purification
through GST-tag column to remove nonspecific binding proteins. The
amount of protein in fraction collected after different washes, (lanes 56) as indicated in the legend, were also analyzed via SDS PAGE on a
15% polyacrylamide gel. The resin (lane 6) was also checked for any
bound proteins that was not eluted from the column to the elution
fraction.
Comparing Positive and Negative Columns. The elution
samples were analyzed via SDS PAGE on a 12% gel. Upper gels
(CBB and silver staining) were SDS/Urea elute sample. Lower gel
(silver staining) was high salt buffer elute sample. Equal protein
amount loaded between Q70L and EM960s sample based on
A280 value. The blue arrows indicate the bands that appear in the
positive control, Q70L column, but not in the negative control,
EM960 column. These bands were not detected in the CBB
staining gel indicating low protein content.
• Q70L, EM960 and Vector columns were both successfully
prepared.
Summary of Events
Centrifuge to collect E.coli
pellet and resuspend in lysis
buffer, followed by cell
disruption by micofluidizer.
Then centrifuge again to
collect the supernatant
containing the GST-fused
Rab5s protein. Filter the
supernatant through 0.2μm
pore filter.
Grew E. coli cells containing
constructs in LB media until
OD600 is around 0.6. Add IPTG
to final concentration of 0.1-1
mM
to express GST-fused
Rab5a proteins.
Check the starting
material, resin,
and flow through
for the column
condition by SDS
PAGE.
De-husk and homogenize 7g
of developing seeds in (3mL
per 1g) CSB buffer per
column. Then centrifuge at
100g for 1min to remove
large starch grain. Then
centrifuge supernatant at
3000 rpm for 10 min to get
pellet. Resuspend the pellet
in CSB buffer containing 1%
Triton X-100.
Spin at 100Kg for 60 min.
supernatant
• To prevent non-specific binding, the rice seed extract was
crudely purified by passing through the GST-tag column. Since
the eluted extract also contained GST-tag protein, future
experiment may require decreased DTT concentration in the
CSB buffer.
Incubate supernatant with
Glutathione Sepharose
resin for 2 hours to create
mutant Rab5a affinity
columns.
• The use of SDS and Urea elution buffer led to the elution of
GST-Rab5as as well, thus, we have to reconsider elution buffer
conditions.
• Comparison of the elution samples by SDS-PAGE of positive
Rab5a column, Q70L, with the negative column, EM960, we
found specific bands in Q70L elute fraction that does not
appear in the negative control, but protein content is too low.
end
To determine effector protein,
cut out specific band in Q70L
elute sample and for tandem
mass spectrometry analysis.
•Optimization of Rab5a affinity chromatography column.
-Check buffer condition.
Pellet: Resuspend
the pellet in CSB
buffer containing
1% Triton X-100,
200mM NaCl. Then
Spin at 100Kg for
60min.
Then dilute with an
equal volume of CSB to
dilute detergent
concentration.
Incubate the Rab5a column with
1mM GTP. Then was wash with NS
buffer (w/o GTP). Mix resin with rice
extract and incubate for 2hours at
4°C on rotating mixer.
Add 0.5 mL Elute
buffer (A/B) and
collect elutant as
sample.
(Elute sample)
Wash with buffer B until
A280 is near zero.
Wash with buffer A until A280
is near zero.
Collect flow through
sample.
This work was supported by the National Science Foundation REU program
under grant DBI-0605016.
1
8
Q70L EM960
start
Rice is the staple food source of nutrients for both humans and
livestock throughout the world. The major proteins in rice seeds
are the storage proteins, glutelins and prolamins. Their mRNAs are
localized at different subdomains of the endoplasmic reticulum
(ER). Glutelin mRNAs are targeted to the cisternal ER, while
prolamin mRNAs are targeted to the protein body ER (PB-ER)
membranes that delimit intracisternal prolamin inclusions. The
glup4 mutant, which causes glutelin mRNAs to be transported to
the PB-ER instead of their normal destination on the cisternal ER,
lacks the small GTPase, Rab5a. In order to find the underlying
cause of this error in mRNA targeting, these studies were
undertaken to identify an associated effector protein of Rab5a that
specifies its role in RNA targeting. We used Escherichia coli to
express GST-tagged-Rab5a positive and negative constructs. Rab5aQ70L, the GTP fixed (active) form, was used as a positive control
while Rab5a-EM960, which has mutation in the effector region
(G45D), was used as a negative control. The purified GST-Rab5a
proteins were attached to Glutathione-linked Sepharose resin to
create an affinity chromatography column. These columns were
used to “fish out” the Rab5a associated effector proteins in rice
developing seed extracts. The eluted proteins from the positive and
negative Rab5a columns were directly compared by SDS
polyacrylamide gel electrophoresis. Initial studies show no
significant differences in the polypeptides eluted from these
columns.
•Tandem mass spec analysis of candidate effector protein
bands if specific bands are presented in Q70L elute fraction.
Check starting material, flow
through, wash A, wash B, and elute
sample fractions via SDS-PAGE.
Summary of Buffers
Lysis buffer
1xPBS, 5mM MgCl2 and 5mM 2-ME,
1mM PMSF
CSB buffer
5 mM Hepes-KOH pH7.5
10 mM MgOAc, 2mM EGTA
1 mM PMSF, 1mM DTT
5 ug/mL Leupeptin (5mg/mL)
1 ug/mL PepstatinA (1mg/mL)
Nucleotide Stabilization buffer (NS buf.)
20 mM HEPES-NaOH pH7.5
100 mM NaCl, 5 mM MgCl2,
1 mM DTT, 1 mM GTP
Wash buffer A
20 mM HEPES-NaOH pH.7.5
100 mM NaCl, 5 mM MgCl2
1mM DTT
Wash buffer B (High Salt)
20 mM HEPES-NaOH pH.7.5
250 mM NaCl, 5 mM MgCl2
1mM DTT
Elution buffer A
0.2% SDS, 4M Urea
Elution buffer B
20 mM HEPES-NaOH pH.7.5
2 M NaCl, 20 mM EDTA, 1mM DTT
•Alternative methods: try pull-down assay or co-IP.
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