Download Proteomics Screening and Identification of Differentially

Biol 475: Microbiology & Biotechnology
Proteomics Screening and Identification
of Differentially distributed membrane
proteins in Escherichia Coli
Erh-Min Lai, Usha Nair, Nikhil D Phadke & Janine R. Maddock
Yehchung Chang
Apurva R Ranka
Question
Which proteins were immunolocalized
and what were they tagged with?
The proteome is defined as the set of all
expressed proteins in a cell, tissue or organism
(Wilkins et al., 1997).
Proteomics can be defined as the systematic
analysis of proteins for their identity, quantity
and function.
What is a minicell?
• Created through cell division in the absence of the Min system
• Composed of two poles without lateral membrane
• Enriched in polar proteins
• Small spherical and anucleate
What is a rod cell?
• Counter part to the minicell
• Enriched in lateral membrane proteins
• Viable and elongated
Differentially distributed membrane proteins in E. coli
• Involved in diverse cellular processes such as
chemotaxis, motility, actin polymerization,
chromosome partitioning and cell division
• Identification through 2-D electrophoresis and mass
spectrometry (MS)
• Analysis of Immunobloting and Immunolocalization
Types of differentially distributed proteins
• Membrane Chemoreceptor Proteins (MCPs)
• Cell Cycle Dependent
• External cues – Yersinia Pestis
• Unknown Polar Protein mechanism
-Actin Polymerization – Shigella Flexneri
-Sporulation – Bacillus Subtilis
1-D SDS-PAGE gel
• Differences in Protein profiles of Rod cells and
Minicells
Membrane protein profiles of rod cells and minicells.
Carbonate-washed membrane proteins from rod cells and
minicells were separated using 12% glycine-SDS PAGE.
Aliquots of 25 µg of proteins from rod-shaped cells (lane 1)
and minicells (lane 2) were loaded on gels stained with
Coomassie blue G-250. For gels stained with silver, 5 µg of
protein from rod-shaped cells (lane 3) and minicells (lane 4)
was loaded. The sizes of molecular weight standards (M)
(Novex unstained marker, Invitrogen) are indicated on the
right in kDa. Arrows indicate differentially enriched bands. For
this experiment, the E. coli minicell mutant X-1488 was grown
at 37°C.
2-D Gel Electrophoresis
• Fractioned using pH 4-7 IPG strips
• Silver staining
Two-dimensional gels of carbonateinsoluble membrane proteins from
minicells (A) and rod cells (B)
fractionated using pH 4 7 IPG
strips followed by 11%
SDS PAGE. Samples of 50 µg of
protein were loaded on each gel.
Proteins were stained with silver.
The pH range is shown at the
bottom, and the sizes of molecular
weight standards (Mr) (Gibco
prestained markers) are indicated
on the right in kDa.
Composite image of 2D- Gel Electrophoresis
Peptide Mass Fingerprinting & Identification
• Protein spots are cut out and washed
• Trypsin Digestion
• Sample Prep and Loading
• MALDI-TOF
• Database Search
-SWISS PROT
-SIGNALP & PSORT
Identification of differentially distributed membrane
Proteins
Minicell-enriched Proteins
• 20 out of 36 identified; 13 Unique
• FtsZ - Most abundant of all cell division proteins
• YbhC; YiaF; OmpW – Strong candidates for septal or polar proteins
• BtuB; AtpA; AtpB; AcrA; FadL – Minicell Bias and DE>1
• OmpT; Tsx; PaI – Multiple isoforms and more enriched in Minicell
• Unidentified
Identification of differentially distributed membrane
Proteins
Rod cell-enriched Proteins
• 13 out of 15 identified; 7 Unique
• NlpB; YfgL; UPO5 – Multiple isoforms and more enriched in Rod Cell
• OstA; OmpX –Rod cell Bias
Special Interest
• OmpA – Many isoforms; Three enriched in Minicell; One enriched in Rod cell
• TolC – Many isoforms; Three enriched in Rod cells; Possible enrichment in
Minicell
Immunoblot Analysis of “Special Interest” Proteins
Inner membrane MCPs
• Predominantly polar and do not resolve well on 2-D gels
•1.5 fold enhancement in Minicell at 30 °C
•5 fold enhancement in Minicell at 37 °C
Membrane proteins from rod cells are loaded in lanes 1 and 3.
Lanes 2 and 4 show membrane proteins from minicells. Cells were
grown at 30°C for samples shown in lanes 1 and 2 and at 37°C for
samples shown in lanes 3 and 4.
TolC
• Majority of isoforms enriched in Rod cells during 2-D gel electrophoresis
•3 to 4 fold enhancement in Rod cells
Immunoblot Analysis of “Special Interest” Proteins
OmpA
• Enrichment in both and also equal distribution in 2-D gel electrophoresis
•Distributed equally between Rod cells and Minicells
Membrane proteins from rod cells are loaded in lanes 1 and 3. Lanes 2 and 4
show membrane proteins from minicells. Cells were grown at 30°C for
samples shown in lanes 1 and 2 and at 37°C for samples shown in lanes 3
and 4.
PaI
• More enrichment in Minicell in 2-D gel electrophoresis
• 1.2 fold enrichment at 37 °C and 30 °C
Construction of E.coli strains encoding Epitope tagged
proteins
• Amplification by PCR of E. coli K-12 protein coding gene
• Digestion with BamHI and cloning into pBluescript SK+
• Verification by sequencing
• Amplification using protein coding gene as a forward primer
and reverse primer containing the epitope tag.
• Cloned into Pcr2.1-TOPO TA cloning vector
• Tagged protein coding gene excised as an restriction enzyme
fragment and then sub cloned into a vector
Localization of membrane proteins by
Immunofluorescence microscopy
OmpW
• C- terminally Fused to Haemagglutinin(HA)
• 35% cells expressed detectable levels with plasmids
• Localized predominantly to the cell poles
• No detection with chromosomes
Localization of membrane proteins by
Immunofluorescence microscopy
YiaF
•C- terminally Fused to FLAG epitope tags
• Patchy localization when expressed from
chromosome or a medium-copy plasmid
OmpA
• Randomly Localized
• Detected using Anti-OmpA antibody
Known or putative function of the identified
Differentially distributed membrane proteins
Protein
na
m
e
Differentially
enriched
compartm
ent
Evidence for
differential
distribution
Known or putative function and localization
MCP
Minicell
Immunoblot analysis
Chemoreceptors, IM
AtpA
Minicell
2-DE comparison
F0F1 ATP synthase α -subunit, IM
AtpB
Minicell
2-DE comparison
F0F1 ATP synthase β-subunit, IM
YbhC
Minicell
2-DE comparison
Hypothetical lipoprotein, pectinesterase family, OM
Tsx
Minicell
2-DE comparison
Colicin K and phage T6 receptor, channel for nucleosides, OM
YiaF
Minicell
2-DE comparison
Hypothetical novel protein, IM
OmpW
Minicell
2-DE comparison
Putative colicin S4 receptor, OM
Pal
Minicell
2-DE comparison Immunoblot analysis
Putative lipoprotein, colicin import, membrane integrity
BtuB
Minicell
2-DE comparison
Vitamin B12 receptor, OM
FtsZ
Minicell
2-DE comparison
Z-ring subunit at cell division sites, IM and cytoplasmic
AcrA
Minicell
2-DE comparison
Component of a multidrug efflux pump, putative IM lipoprotein
FadL
Minicell
2-DE comparison
Long-chain fatty acid transport protein, OM
OmpT
Minicell
2-DE comparison
Outer membrane protease
TolC
Rod-shaped cell
2-DE comparison
Immunoblot analysis
Multidrug efflux, protein export, tolerance to colicin, cell division, OM
YfgL
Rod-shaped cell
2-DE comparison
Putative lipoprotein, OM or IM
NlpB
Rod-shaped cell
2-DE comparison
Putative lipoprotein, OM
UPO5
Rod-shaped cell
2-DE comparison
Hypothetical outer membrane protein
OstA
Rod-shaped cell
2-DE comparison
Involved in outer membrane biogenesis, OM
Discussion
• 12 Polar membrane proteins that are differentially enriched.
• Not all membrane proteins differentially enriched were identified
• Immuno-localization confirms Proteomics analysis
- OmpW (Outer membrane protein)
- YiaF (Inner membrane protein)
• Differences in protein and lipid composition that contribute to the
different micro environments of lateral and polar membranes
-PaI ; YbhC ( Minicell enriched)
-YfgL; NlpB (Rod Cell enriched)
Discussion (contd)
• Peripheral membrane proteins of the F0F1ATP synthase complex
detected but membrane embedded proteins of the complex not
detected
- AtpA; AtpB (Associated with extra-membranous F1)
- AtpF (Associated with membrane embedded F0)
- Unable to detect YidC (Important for membrane insertion of F0)
• Porin like outer membrane proteins are enriched in minicell
membranes.
- Tsx (ColicinK receptor, Channel for nucleoside import )
- OmpW(Colicin S4 receptor)
- BtuB (Binding and Uptake of B12 and bacteriophage BF23)
Discussion (contd)
• YiaF three fold more abundant in minicell and resolved as a
single spot.
- Hypothetical inner membrane protein
- Highly conserved in E.coli; S.flexneri; Salmonella enterica
- Homology in Y. Pestis; Pseudomonas syringae
- Disruption did not reveal growth phenotype
• TolC has the possibility of having two different isoforms with
different spatial localization
- Production of peptide antibiotic microcin; secretion of colicinV
- Outer membrane integrity; chromosome segregation
Proteomics as a resource
• Identification of proteins
• Initial step in dissecting protein micro-environment in a cell
Future Challenges
• Localizing identified proteins
• Assessing specific roles
Issues
• Good Overview
• Some data not shown
• Should have addressed the negative staining in SDS-PAGE in
detail