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Detection analysis of the DEAD-box protein DBP3 in budding yeast
Biol110L Cell Biology Laboratory; Thimann Labs 203; Molecular, Cellular &
Developmental Biology Department, UC Santa Cruz, CA 95064
One of the most distinct characteristics of eukaryotic cells is the compartmentalization of
various proteins. Here we have characterized cellular association and topology of green
fluorescently tagged DBP3 protein of the DEAD-box family in yeast cells. In order to do
so, cellular compartmentalization of the fusion protein was determined by microscopy
and differential centrifugation. Additionally, the topology of our fusion protein was
determined by biochemical fractionations. Identification of our fusion protein was then
detected using immunoblotting techniques. Analysis of results characterized the DBP3
protein as a peripheral protein associated with the nucleolus of yeast cell.
Introduction
One of the main goals of cell biology is to understand cells’ biological properties
including understanding protein’s function. An important characteristic of eukaryotic
cells are the complexity of their cellular structure and compartmentalization.
Understanding how proteins are organized and compartmentalized within the cell is
detrimental to understanding how proteins are regulated.
Originally used for baking and brewing, yeast have long developed into
organisms ideal for studying cell compartmentalization and protein regulation. Many
scientists have since then used the yeast Saccharomyces cerevisiae as model
eukaryote organism. To determine where a certain protein is located within a cell, we
fluorescently tagged a DBP3 protein, a DEAD-box protein with green fluorescent protein
(GFP). Using this strategy we were able to follow the fate of the DBP3 protein from its
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associated compartment, the nucleolus
without disrupting cell integrity. The
fluorescently tagged proteins will help us visualize the location of the protein is using
fluorescent microscopy. We will then be able to characterize the fusion protein by
western blot.
Found in both prokaryotes and eukaryotes, DEAD-box proteins play an integral
role in RNA metabolism and processing (2). Previous sequence analysis revealed that
in S. cerevisiae, about only 2% of the protein-encoding genes code for DEAD-box
proteins. DBP3 is particular, was found to be associated with the nucleolus and nuclear
outer membrane where its main biological functions involves ribosome biogenesis (1).
Its molecular function was characterized to as being a putative ATP-dependent RNA
helicase. DBP3 has been characterized with having nine conserved motifs, forming a
pocket to bind ATP and RNA, where it is able to unwind RNA strain by hydrolyzing ATP
(2).
In order to characterize DBP3 protein localization, DBP3 protein was
fluorescently tagged and visualized using light and fluorescent microscopy. To further
characterize the location of our fusion protein of interest, differential centrifugation was
conducted, where cell fractionation was subjected to various velocities in attempt to
isolate the location of our fusion protein. To determine the membrane topology of our
fusion protein, various biochemical fractionations were conducted and visualized
through western blot.
Materials and Methods
Preparation of yeast strain
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Yeast strain used in this study, YS8 contained GFP tagged DBP3 protein was
stored in -70°C prior to inoculation. YS8 strain was then inoculated into liquid media
containing 1x YP with 5% glucose (YPD media). Yeast strain was then grown at 30°C
in a floor incubator for two days.
Visualization of yeast strain using light and fluorescent microscopy
Liquid cultures of YS8 strain were grown in YPD liquid media and 200µl of liquid
cell culture were separated into two tubes (~1 OD600 and 1.4E7 cells each). A total of
2.8E7 cells were harvested by centrifugation at 5,000 rpm for 1 minute. In each
microcentrifuge tube, 10µl of minimal media with 2% glucose were added or with 20%
1,6 hexanediol (HD). 1.75µl of each culture were placed onto a microscope slide and
light and fluorescent image of the yeast cells were taken.
Preparation of yeast spheroplasts
In order to gain access to the GFP-tagged protein of interest, yeast’s cell walls
were removed, yielding yeasts spheroplasts. Prior to cell harvesting, cells were grown in
400 ml of 1x YP media with 5% glucose at 30°C. Cells were grown until log phase of
growth and cell density reaches 2 OD600/ml.
A total of 1.084 OD600 cells were harvested from 400 ml of culture media by
centrifugation in a Sorvall GSA rotor at 5,000 rpm for 5 minutes at 4°C. Cells were
resuspended in high-pH buffer of 100mM Tris pH 9.4 and fresh 10mM DTT to a
concentration of 50 OD600/ml. Cells were then incubated in room temperature for 5
minutes and then sedimented at full speed for 5 minutes in room temperature. Cells
were then resuspended in 43.36 ml of spheroplasting medium (0.75x YP, 10mM Tris pH
7.5, 0.7 M sorbitol, and 0.5% glucose) to a target concentration of 25 OD600/ml. Cell
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density (OD600/ml) of a 1:100 dilution was measured and lyticase enzyme was added
(15µl for every 25 OD600/ml) and incubated at 30°C in a shaking incubator. Cell density
of a 1:10 dilution was measured at 5 minute intervals until cell density of the 1:10
dilution reaches approximately 10% of the original starting value.
Yeast cells were stored on ice for 2 minutes before centrifuged at 2,000 rpm for 5
minutes in Sorvall SA600 rotor. Cells were resuspended in 10ml of regeneration
medium (0.75x YP, 0.7 M sorbitol, and 1% glucose) and incubated in a shaking
incubator for 30 min at 30°C. Cells were then sedimented at 5,000 rpm for 5 min in 4°C
in a Sorvall GSA rotor before resuspending cells in a chilled iso-osmotic buffer (20 mM
Hepes pH 6.8, 400mM sorbitol, 150 mM KOAc, 2 mM Mg(OAc)2, and 0.5 mM EGTA) to
100 OD600/ml. Yeast cells were sedimented at 5,000 x g for 5 min at 4°C in a SA600
Sorval rotor and then resuspended in an iso-osmotic buffer to a final concentration of
300 OD600/ml. 200 µl of the suspension were aliquoted into 10 microcentriuge tubes and
stored in -70°C.
Cellular fractionation and differential centrifugation
From previous aliquot, 10 µl was saved as pre-lysis sample saved on dry ice and
500 µl of chilled iso-osmotic buffer was added to the remaining aliquot. Spheroplasts
were sedimented at 5,000 rpm for 30 sec before being resuspended in 500 µl of chilled
iso-osmotic buffer. Spheroplasts were sedimented as previous. Spheroplasts was
resuspended with 400 µl of low-osmotic support buffer (20 mM Hepes pH 6.8, 50 mM
sorbitol, 50 mM KOAc, 2 mM Mg(OAc)2, and 0.5 mm EGTA) and 50 µl of high-osmotic
support buffer (20 mM Hepes pH 6.8, 1.2 mM sorbitol, 600 mM KOAc, 2 mM Mg(OAc)2,
and 0.5 mm EGTA). 25 µl was saved as our whole cell extract (WCE) and remaining
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spheroplasts was sedimented 2,000 rpm for 2 min. Obtained supernatant was saved as
our low speed supernatant and remaining pellet was resuspended with 450 µl of isoosmotic buffer. 25µl of this suspension was saved as our low speed pellet (LSP). Low
speed supernatant was centrifuges at 13,000 rpm for 10 min and medium speed
supernatant (MSS) was saved. Remaining pellet was resuspended with 200 µl of isoosmotic buffer and 10 µl was saved as our medium speed pellet (MSP). 200 µl of the
medium speed supernatant was centrifuged at 250,000 x g for 10 min at 4°C using
TLA100.2 rotor and the high speed supernatant (HSS) was collected. The remaining
pellet was resuspended with 20 µl and was saved as our high speed pellet (HSP). All
saved samples were stored in dry ice.
Preparation of yeast spheroplasts for protein topology studies
Frozen spheroplast aliquots was thawed and 500 µl of chilled iso-osmotic cuffer
was added and centrifuged at 5,00 rpm for 30 sec. spheroplasts was resuspended in
500 µl of chilled iso-osmotic buffer and sediment cells as previous. Cell pellet was
resuspended with 400 µl of low-osmotic support buffer and incubated for 90 sec before
adding 50 µl of high osmotic support buffer. 100 µl spheroplasts suspension were
aliquoted into 4 microcentrifuge tubes each containing 25 µl of: iso-osmotic buffer, 5M
NaCl, 10% Triton X-100, or 75% 1,6 hexanediol. After incubation on ice for 5 min, tubes
were centrifuged at full speed for 10 min and 100 µl of supernatant from each was
collected and stored on dry ice for biochemical fractionation studies.
SDS-PAGE
25 µl of WCE and 25 µl LSP were combined with 225 µl of 2x Laemmli sample
buffer (0.3M Tris pH 6.8, 36% glycerol, 10% SDS, 0.012% bromophenol blue (with β-
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mercapethanol (1:50 dilution) and protease inhibitors). 20 µl of HSP and 10 µl medium
MSP were combined with 20µl 4x Laemmli buffer. For biochemical fractionations, 20 µl
MSS was mixed with 4x Laemmli buffer. Various protein samples mixed with Laemmli
buffer was heated at 90°C in a heat block for 10 min. 25 µl of each samples were
resolved in an 8% polyacrylmide gel for 60 min at 20 mA. Proteins were then transferred
to PDVF membrane by electrophoresis at 50V for 2h.
Western blot and Amido black protein staining assay
PDVF membrane was rinsed with water and stained with amido black ink solution
(amido black in 10% acetic acid) and destained using 10% acetic acid destaining
solution. Membrane was then blocked with TBST with 5% milk for 30 min and incubated
with primary antibody solution (rabbit anti-GFP) for 30 min. Membrane was rinsed 3x
with TBS-T with milk and incubated with secondary antibody (donkey anti-rabbit) for 30
min. Membrane was then rinsed 3x with TBS-T with milk and membrane was then
incubated with 15 chemiluminescent solutions A and B provided by the lab. Membrane
was dried and exposed to X-ray film for 10 sec.
Results
Sequence analysis
In order to characterize our GFP tagged DBP3 protein, known amino acid
sequence (Fig. 1A) was run through BLASTP using the saccharomyces genome
database. Results characterized the DBP3 protein to be an RNA helicase belonging to
the DEAD-box family protein associated with the nucleolus. The predicted molecular
weight of the GFP tagged DBP3 protein was determined to be 88 kDa based on known
amino acid sequence of the protein (Fig. 1A).
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In order to predict if our protein of interest, DBP3 has membrane-spanning
domains, a Kyte-Doolittle hydrophobicity plot was constructed. We expected the
presence of transmembrane spanning domain if there are at least 20 hydrophobic
amino acids in a row capable of spanning cell membrane. Results indicate the absence
of membrane spanning domains in DBP3 protein (Fig. 1B).
Cellular localization of DBP3
In order to characterize the location of our GFP tagged DBP3 protein, light and
fluorescent microscopy was used to determine localization of DBP3 protein in presence
of minimal media and aliphatic alcohol. Since the DBP3 protein was characterized to be
associated with the nucleolus, fusion protein was expected to localize to the nucleolus.
Figure 2A shows results from fluorescent microscopy, which bright green spots are
seen to localize at a specific location within the cell, presumably the nucleus which are
circular in shape. Since the nucleolus inside a yeast cell was determined to be crescent
moon shaped, we expected to visualize the fluorescent image to be crescent moon
shaped as well. Regardless of the deviations, we still conclude that our fusion protein to
be localized to the nucleolus.
Addition of HD allowed us to visualize the affects that aliphatic alcohol had on our
GFP tagged protein. Results obtained from fluorescent microscopy showed that DBP3
went from a localized state in the nucleolus to being homogeneously distributed
throughout the cytoplasm (Fig. 2B).
Fusion protein was also observed inside the
vacuole, suggesting that HD somehow disrupted the integrity of our protein, which the
cell did not recognize it as a natural protein part of the cell anymore (Fig. 2B).
Cellular fractionation of fusion protein
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In order to further analyze the association of GFP-DBP3 protein in yeast
spheroplasts, cell components were purified by differential centrifugation in order to
determine where our GFP-DBP3 protein fractionates. Proteins extracted from various
fractionations were analyzed by SDS-PAGE.
Prior to immunodetection of GFP-DBP3 protein, amido black staining was
performed for the detection of total amount of protein transferred. Results indicated
successful protein extraction and loading with the exception of the HSP (Fig. 3A). Lane
containing MSP, showed high concentration of proteins loaded, which was determined
by darker protein bands observed in the lane versus the lane containing WCE. Lane
containing HSP was observed to have no bands, which we would have observed if
proteins were present.
Biochemical extracts showed successful transfer and
extractions of proteins, suggested by the presence of dark protein bands in each lane
(Fig. 3A).
Immunodetection analysis of DBP3 using western blot
In order to determine what cellular component DBP3 was associated with,
immunodetection was performed on various fractionations. Results indicate that our
fusion protein was associated with large cell components such as the nucleus, which we
have expected (Fig. 3B). Results also indicate a darker band in the MSP, indicating that
our protein was also associated with medium sized cell component (Fig. 3B). We were
not expecting the presence of our fusion protein in the HSP, because we expected
DBP3 to be associated more with bigger structure such as the nucleus and nucleolus.
We were not able to visualize this, because our sample did not contain any indication of
proteins (Fig.3A).
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In order to characterize protein topology, biochemical fractionation using aliphatic
alcohol (HD), non ionic detergent (Triton X-100), high salt (1M NaCl), and iso-osmotic
buffer was performed. The expected molecular weight of our GFP-DBP3 protein was 88
kDa and all bands observed from gel electrophoresis showed bands located around 100
kDa, which is close enough in proximity with the expected band size (Fig. 3B).
In the presence of high salt (1M NaCl), we expected to visualize a band darker
than one would observe in buffer if the protein was peripherally attached to the
membrane. A darker band was observed where fractionation was exposed to high salt
in contrast to the lighter band observed with buffer (Fig. 3B). This may indicate that our
GFP fusion protein is peripherally bounded to its cellular component.
In the presence of non-ionic detergent, we expected a darker band than the once
observed in buffer if protein was integrally attached to the membrane. We observed, a
band with similar darkness with fractionation exposed to buffer was observed (Fig. 3B).
This may suggest that DBP3 was not integrally bounded to the cell component.
Similarly, fractionation exposed to 15% HD showed similar results as in high salt (Fig.
3B).
Slight anomalies was observed in which heavier bands were seen for every
fractionation reaction exposed to buffer, high salt, non-ionic detergent, and aliphatic
alcohol solution, which may suggest post-translational modification, such as
phosphorylation. We found that this was not the case, because upon further analysis
using
the
online
program
phosphoGRID,
DBP3
does
not
contain
possible
phosphorylation sites for post-translational modification. We thus are unable to explain
the slight anomaly.
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Discussion
Our initial study was to determine the cellular localization of the GFP tagged
DBP3 protein in yeast and characterize the protein by biochemical fractionation by
differential centrifugation. Given its function as being an RNA helicase and assisting in
ribosomal biogenesis, we expected the protein to be associated with the nucleus and/or
nucleolus and act as peripherally attached protein due to the absence of
transmembrane domain.
Using light and fluorescent microscopy, we cannot determine with certainty if our
protein was actually associated to the nucleus or nucleolus, due to poor resolution.
Although microscopically we were unable to clearly visualize the exact location, DBP3
has been known to aid in ribosomal biogenesis, which occurs mainly in the nucleolus of
yeast cells. We expected to visualize a crescent shape image, which is the shape of the
nucleolus in yeast. We observed a slightly different image upon the analysis of our
fluorescent microscopy pictures, which was a circular shape rather than the expected
crescent moon shape (Fig.2B). This deviation in our observation can simply be due to
the microscope being too bright, the coverslip may be too thick or too much oil causing
more diffraction contributing to the circular shape. Another possible explanation might
be the fact that we obtained yeast sample from a liquid culture, where perhaps cells are
more active and thus contributes the blurry circular image. Perhaps if the cells were
obtained from colonies grown on a plate, cells would be more stationary and a clearer
image of the crescent moon shape nucleolus would have been seen.
To determine the effects of aliphatic alcohol on protein localization, we
suspended yeast spheroplasts in HD. Other than knowing that HD was a small
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hydrophobic molecule, we had no expectation on its effects on DBP3 protein
localization. Interestingly, we observed that cell integrity was maintained (Fig. 2B).
Being a small hydrophobic molecule, HD was able to diffuse through the plasma
membrane without disrupting cell shape and perhaps effect protein interaction with
internal membrane. This conclusion was derived from the observation of protein
homogeneously distributed throughout the cytosol, but void of the vacuole. This may
suggest that HD might not have affected protein structure so much that the cell
recognizes it as foreign protein and designates it to be destroyed.
Since we expected the DBP3 to be localized mostly in the nucleus or nucleolus,
we expected our protein to fractionate with the LSP and MSP as seen by
imuunodetection (Fig. 3B). A band present in the LSP suggest that out protein was
attached to something large, such as the nucleus which we expected as well, because
DBP3 is known to be associated with the nucleus in addition to the nucleolus (Fig. 3B).
Since DBP3 was known to be mostly being associated with the nucleolus, we
expected larger protein concentration in the fractionation containing MSP. We observed
that more of our protein fractionated with the MSP, which the nucleosome can be found.
A darker band was seen in MSP perhaps due to a higher concentration of protein
loaded, which can be seen by dark bands observed in the amido black stain (Fig. 3A).
We also observed that our protein was not present in the high speed supernatant
(HSS), which suggests that the DBP3 protein was not free floating and that it was
bounded to the nucleolus.
In order to characterize the topology of our protein, we performed biochemical
fractionation of our fusion protein. Based on our hydrophobicity plot, we expected our
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protein to be peripherally attached to the membrane, due to the absence of
transmembrane spanning regions (Fig. 1A). Upon the exposure to high salt, we
expected the dissociation of our peripherally attached protein from the membrane. As
expected, we observed a dark band where salt was added to MSS in contrast to the
sample added with buffer (Fig.3B). We concluded that our protein was peripherally
attached to an organelle that is medium sized. This does only suggest that our protein
was bound to a membrane, but to non-membranous organelles also such as the
nucleolus. Fractionation with Triton X-100 resulted in a band with the same color
intensity as the one in buffer, confirming that our protein was not an integrally bounded
protein (Fig.3B). This further confirmed that our protein was a peripheral protein. We
also observed the effects of aliphatic alcohol by using HD. A dark was also observed
when exposed with HD, with the same color intensity when fractionation was exposed to
high salt (Fig. 3B). This suggests that HD somehow affects protein interaction by
dissociating peripherally bounded proteins from the membrane. Overall, based on our
results, we can safely conclude that the DBP3 protein was associated with the
nucleolus and was a peripherally attached protein. For future research, perhaps one
can further characterize DBP3 protein by analyzing gene expression and track its fate
from initial assembly to the final location where the protein performs its main function.
With the consistent need to understand the way our cells work and how proteins
play such integral roles in cellular function, out study provides possible methods
towards characterizing different proteins by tagging GFP to our protein of interest.
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Figures
A
PEPTSAVASEFYVQSEALTSLPQSDIDEYFKENEIAVEDSLDLALRPLLSFDYLSLDSSIQAEISK
FPKPTPIQAVAWPYLLSGKDVVGVAETGSGKTFAFGVPAISHLMNDQKKRGIQVLVISPTRELA
SQIYDNLIVLTDKVGMQCCCVYGGVPKDEQRIQLKKSQVVVATPGRLLDLLQEGSVDLSQVNY
LVLDEADRMLEKGFEEDIKNIIRETDASKRQTLMFTATWPKEVRELASTFMNNPIKVSIGNTDQL
TANKRITQIVEVVDPRGKERKLLELLKKYHSGPKKNEKVLIFALYKKEAARVERNLKYNGYNVAA
IHGDLSQQQRTQALNEFKSGKSNLLLATDVAARGLDIPNVKTVINLTFPLTVEDYVHRIGRTGRA
GQTGTAHTLFTEQEKHLAGGLVNVLNGANQPVPEDLIKFGTHTKKKEHSAYGSFFKDVDLTKK
PKKITFD
B
Figure 1. (A)Amino acid sequence of GFP tagged DBP3 protein. Molecular weight of the
protein was analyzed using ExPASy’s ProtParam tool and was determined to be 88
kDa. Sequence analysis and protein characterization was determined by running the
sequence through BLASTP program. (B) A Kyte-Doolittle hydrophobicity plot of the
DBP3 protein was constructed in attempt to determine the presence of membrane
spanning domain. A window size of 20 was chosen to make hydrophobic, membranespanning domain predominant. Results indicate the absence of membrane spanning
domains.
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A
B
Figure 2.Yeast strain, S.cerevisiae were grown in liquid media containing 1x YP with 5%
glucose (YPD media). Yeast strain was then grown at 30°C in a floor incubator for two
days until log phase of growth. Cells were harvested by centrifugation and protein
localization were visualized using light and fluorescent microscopy using 100x objective.
Yeast spheroplasts were suspended in minimal media to determine localization of the
fusion protein. Looking at the merged image, one is able to determine that the GFPDBP3 protein was localized in the nucleolus. In addition, yeast spheroplasts were also
exposed to aliphatic alcohol (15% HD) to determine the effect the compound has on
protein localization. Merged images in panel B showed homogenous dispersion of the
protein throughout the cytosol.
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Figure 3. Observation of GFP tagged DBP3 protein in various fractionations extracted
using differential centrifugation. (A) Various protein fractionations and biochemical
fractionation was first mixed with Laemmli buffer (0.3M Tris pH 6.8, 36% glycerol, 10%
SDS, 0.012% bromophenol blue (with β-mercapethanol (1:50 dilution) and protease
inhibitors). Polyacrylmide gels was then stained using amido black staining solution
(amido black in 10% acetic acid). All lanes with the exception of the high speed pellet
(HSP) showed successful extraction and transfer of proteins. Note that lane with
medium speed pellet (MSP) contained a higher amount of protein loaded into the well
than in well containing whole cell extract. Biochemical fractionation using buffer, 1M
salt, 2% triton-100, 15% 1,6-hexanediol showed positive extraction of proteins from their
cellular compartment. (B) PDVF membrane containing fractionations was blocked with
TBST with 5% milk and incubated with antibody probes. Membrane was dried and
exposed to x-ray film for 10 sec. Protein bands were observed in WCE, LSP, and MSP
to be around 100 kDa. Darker bands were observed in fractionations added with
aliphatic alcohol and 1M salt versus the lane containing buffer.
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References
(1) Garcia, Ivelitza, and Olke Uhlenbeck. "Differential RNA-Dependent ATPase
Activities of Four rRNA Processing Yeast DEAD-box Proteins." Biochemistry 47.47
(2008): 12562-12573.
(2) Linder, Patrick. "Dead-box proteins: a family affair—active and passive players in
RNP-remodeling." Nucleic Acids Research 34.15 (2006): 4168-4180.
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