Transcript Document

Global Analysis of Protein Localization in Budding Yeast
Won-Ki Huh, James V. Falvo, Luke C. Gerke, Adam S. Carroll, Russell W. Howson, Jonathan S. Weissman & Erin K. O’Shea
Presented by Ji-eun Lee and Teresa Tan
Microbiology 475
December 3, 2003
Article Question:
You are a researcher trying to determine the subcellular
localization of a protein in yeast but, much to your dismay,
you find that you are not able to assign the protein to a
localization category by GFP microscopy alone. What
experiment could you perform to determine which subcellular
compartment the protein localizes to in yeast?
Answer:
Use co-localization (with proteins of known localization
labeled with a different marker).
Background
GFP (green fluorescent protein) found in
Aequorea victoria, first discovered in 1960’s
•Mechanism: spontaneous fluorescence,
“can” structure, excitation peak at 508 nm
•Uses: in-vivo marker of protein
localization
•Advantages: patterns of protein
expression intact, no external co-factors,
may be monitored in living cells
•Disadvantages: mislocalization through
C-terminal fusion
ORF (open reading frame) may potentially encode part or all of
a protein
Fluorescence Microscopy Uses: detect structures, molecules,
proteins within the cell; fluorescent molecules (i.e., GFP)
absorb light at one λ and emit at a longer λ
Purpose of This Study
To analyze cellular compartment localization of proteins
in budding yeast, Saccharomyces cerevisiae, in order to
better understand the proteins’ functions
To utilize GFP fusion proteins and fluorescence microscopy to
analyze subcellular protein localization
To understand relationship between genetic and physical
interactions and subcellular localization to
predict/corroborate protein functions
Methods
Construct GFP-tagged library:
6,234 ORFsPCR6,029 GFP-tagged ORFs
Fluorscence Microscopy4,156 total ORFs
GFP-tagged Library Construction
Fusion protein: homologous recombination of GFP with ORF
at the C-terminus
GFP fluorescence microscopy: 4,156 ORFs
showed meaningful GFP signals
Methods Continued
Co-localization experiment: to refine the 12 original
localization categories
mRFP: monomeric red fluorescent protein strains
fused to proteins whose localization are known
(reference proteins) were mated with 700 GFP strains
whose localizations are unknown (see Figure 2c)
An additional 11 localization categories were generated to
account for the proteins whose localizations were
previously unknown (see Table 2)
Co-localization Micrographs
GFP + RFP fusion strains were mated and their
micrographs merged
12 subcellular locations initially classified (left
column)
Additional 11 localization categories defined via
co-localization (right column)
Subcellular Localization of Yeast Proteins
4,156 proteins represented 75% of yeast proteome
Localization data accounted for 70% of previously
unlocalized yeast proteins
56%: nucleus & cytoplasm; 44%: other subcell. locations
80% agreement with Saccharomyces Genome Database
(SGD) for 2,526 proteins
29 nuclear pore complex proteins identified by Mass
Spectrometric analysis, 25 were visible
Top: authors’ contribution to localization data for yeast proteome
Bottom: distribution of subcell. localizations for proteins
Nucleolar Protein Localization
Authors provided 1st direct evidence for some nucleolar
proteins that they reside in nucleolus
164 proteins in nucleolus82 overlap with SGD’s 127
which means 82 are newly defined nucleolar proteins
Mass Spec: 271 human nucleolar proteins, 166 homologues in
yeast (authors detected 164 yeast nucleolar proteins)
Expect human homologues of yeast proteins detected in
nucleolus will also be nucleolar proteins
Authors’ contribution: 82 newly defined nucleolar
proteins
Classification of the authors’ 164 identified nucleolar
proteins
Protein Localization and mRNA Co-expression
Compared transcriptional co-regulation to subcellular protein
localization on a proteome-wide scale
Utilized previous study that identified 33 transcriptional
“modules” – genes with marked co-regulation
Definition of enrichment: ratio of fraction of proteins in each
module with given subcellular localization to that fraction
in whole proteome
19 of 22 most highly expressed modules show statistically
significant enrichment
Strong correlation between co-localization and transcriptional
co-regulation, and furthermore, biological function
Sub-division of sets of co-expressed proteins
Co-localization of co-expressed proteins allows for prediction
for proteins of unknown biological function
Comparison with Genetic and Physical Interactions
Investigated relationship between co-localization and genetic
and physical interactions
Reference set: sum of all genetic and protein-protein
interactions reported in GRID database
Determined fold enrichment of interactions between
localizations
 Compared distribution of subcellular localization of
interacting protein pairs to that of random protein pairs
Strong enrichment of interaction between proteins that co-localize
Degree of enrichment varies widely by compartment (cytoplasm vs.
microtubule)
Known or predicted function of proteins consistent with localization
(grey: previously thought not to localize to these
compartments)
Network of statistically significant interactions connects
functionally and physically related subcellular regions
(e.g. secretory pathway compartments)
Conclusions
Localized 4,156 proteins (75% of yeast proteome)
Provided location for 70% previously unlocalized yeast
proteins (30% of yeast proteome)
Overcame limits of fluorescence microscopy via colocalization of unknown proteins with proteins of
known location fused to another marker
Demonstrated higher frequency of co-localization for
proteins of transcriptional co-expression (“modules”)
Showed significant correlation between interaction and colocalization
Study provides most complete picture of yeast protein
localization to date (Wohlschlegel, J. & Yates, J.)
Future Studies
Organellar proteomics: nucleolar proteins involved in cell
cycle and gene regulation and the authors generated 82
newly defined nucleolar proteins, thus it would be
interesting to investigate their particular functions.
Study global dynamics of yeast proteome under different
external stimuli or growth conditions
Assay effects of deletion or mutation of a protein of
interest on global protein localization
Critiques
The authors point out that proteins may occupy the nucleolus
transiently but do not clarify why proteins leave and where
do they go? If their method of detection is not sensitive
enough, what adjustments would they make to enhance
sensitivity?
Mislocalizations may occur from C-terminal fusion of GFP to
ORF through steric hindrance or interruption of C-terminal
localization sequences
Limitations of fluorescence microscopy: kinetochore vs.
spindle pole body; membrane vs. lumen for mitochondria
or endoplasmic reticulum
Must balance validity of authors’ results (i.e., 82 newly defined
nucleolar proteins) with limitations of f.m. & 2 micrograph
scorers by conferring with localization data obtained by
other methods (i.e., mass spec & other databases)
References
Andersen, J.S. et al. Directed proteomic analysis of the human nucleolus. Curr. Biol.
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Bergmann, S., Ihmels, J. & Barkai, N. Iterative signature algorithm for the analysis
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Breitkreutz, B.-J., Stark, C. & Tyers, M. The GRID: the General Repository for
Interaction Datasets. Genome Biol. 4, R23 (2003)
Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl Acad. Sci.
USA 99, 7877-7882 (2002).
Dolinski, K. et al. Saccharomyces Genome Database
(http://www.genome.stanford.edu/Saccharomyces) (2003).
Ihmels, J. et al. Revealing modular organization in the yeast transcriptional
network. Nature Genet. 31, 370-377 (2002)
Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509-544
(1998).
Wohlschlegel, J. & Yates, J. Proteomics: where's Waldo in yeast? Nature 425, 671-672
(2003).
http://notes.cc.sunysb.edu/CAS/chemistry.nsf/pages/Tonge_Group