Assigning Function to Yeast Proteins

1
Assigning Function to Yeast Proteins By
Integration of Technologies
Hazbun, TR et al. (2003) Mol. Cell 12, 1353-1365.
Kate Drahos 23 April 2004
2
Overview

• Although the yeast genome is fully sequenced,
• 1/3 of ORFs are not functionally annotated
• Large-scale studies provide incomplete data
• Small-scale studies are quite directed and
  provide
• biased information
• Further analysis is required for complete
  functional
• annotation of the yeast genome

3
Goals

• Set of 100 essential ORFs whose functions are
• unknown
• Protein products of ORFs subjected to a set of
  four
• different experimental approaches
• 1) Tandem Affinity Purification MudPIT
• 2) Yeast Two-Hybrid Screen
• 3) Fluorescence Localization
• 4) Protein Structure Prediction
• Aim to assign roles to these proteins based on
• standardized GO categories

4
Tandem Affinity Purification

• TAP used to purify target ORFs and
• copurifying proteins
• TAP allows for native expression levels of the
  tagged
• protein and purification of unknown protein
  complexes

Proteins visualized by SDS-PAGE and silver
staining
(adapted from Rigaut et al., 1999)
5
Multidimensional Protein Identification Technology

• MudPIT developed for analysis and identification
  of proteins
• from purified complexes
• Resolves larger number of peptide fragments than
  traditional
• LC-MS/MS systems by combining strong cation
  exchange
• (SCX) and reverse phase (RP) columns

• Peptides identified by
• electrospray ionization (ESI)
• mass spectrometry

(adapted from McDonald et al., 2002)
6
Yeast Two-Hybrid Screens
Gal4 Binding Domain
ORF
6000 genes
Gal4 Activation Domain
Expression of reporter gene indicates interaction
between the two gene products
6000 genes
ORF
Gal4 AD
Gal4 BD
DNA
Reporter Gene
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Fluorescence Localization

• Provides information on where unknown proteins
  function
• in the cell
• ORFs were tagged with Venus yellow fluorescent
  protein
• YFP-fusions must maintain cell viability

ORF
Venus
8
Protein Structure Prediction

• Ginzu, an iterative process, was used for domain
  parsing
• and structure prediction
• PSI-BLAST identified remote homologies and fold
• recognition servers identified further matches
• ROSETTA was used to predict structures of
  unmatched
• domains and these were compared to known
  structures
• Combinations of these data were used to increase
  
• confidence of structure predictions

9
Gene Ontology Consortium

• GO is a collaborative effort to develop
  consistent
• descriptions of gene products among databases
• Three vocabularies are under construction to
  describe
• ORFs in terms of
• 1) biological process
• 2) cellular localization
• 3) molecular/biochemical function
• Availability of standardized terms should
  simplify
• part of the research process
• www.geneontology.org

10
YDR288w YML023c DNA repair complexes

• Both complexes copurify with Smc5-Rhc18
• YDR288w purified with Nse1, Mms21, and Qri2
  confirmed by
• two hybrid screen
• NSE1, SMC5, and MMS21 mutants show sensitivity
  to DNA
• damaging agents
• YDR288w localized to nucleus

(Hazbun et al., 2003)
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YDR288w YML023c DNA repair complexes

• YML023c purifies with Kre29 confirmation by
• two hybrid screen
• YML023c also localized to nucleus
• YML023c purifies with other DNA repair-related
  proteins
• such as Smt3 and Pac1

(Hazbun et al., 2003)
12
YKR079c DNA/RNA catabolism

• YKR079c purifies with YMR099c and Nuc1
• It localized in two distinct regions in the
  nucleus
• with YMR099c and in the mitochondria with Nuc1
• Nuc1 plays a role in DNA/RNA catabolism and
  exhibits both
• ribonuclease and deoxyribonuclease activities
• YKR079c structure predictions supports its role
  in
• nucleic acid catabolism

(Hazbun et al., 2003)
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YLR424w, YKR022c,YLR132c novel splicing proteins

• YLR424w and YKR022c purify with 18
• spliceosome components
• They also show interaction with each other
  through
• two-hybrid screens
• Both localized to the nucleus
• YLR424w is predicted to have a G-patch domain,
• which is involved in RNA binding
• YLR132c appears to be bifunctional, playing
  roles in
• mRNA splicing and aerobic respiration

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DNA Repair Networks
This suite of protein technologies is a powerful
tool for identification of functions of unknown
proteins and prediction of certain networks of
interactions
(Hazbun et al., 2003)
Strong evidence that the uncharacterized
proteins YDR288w and YML023c are components of
two related DNA repair complexes
15
Summary of Screening

• 77 ORFs annotated with at least 1 GO term
• 48 ORFs annotated with at least 2 GO terms
• 17 ORFs annotated with all 3 GO terms

• Fluorescence localization assigned 63 cellular
• component terms
• TAP/MudPIT assigned 32 biological process terms
• Two-hybrid screen assigned 16 process terms
• Protein structure prediction and homology
  searches
• assigned molecular function terms for 27 ORFs

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Conclusions

• Integration of technologies directly assays a
  selected
• group of proteins for function
• Previous studies that do not combine
  technologies
• identified some, but not all, of these
  interactions
• Integrated approaches can yield large amounts of
  
• information for uncharacterized groups of
  proteins
• Modification of current protocols, or addition
  of new
• approaches should accurately predict the roles
  of many
• more uncharacterized ORFs

17
References
Ashburner, M, Ball, CA, Blake, JA, Botstein, D,
Butler, H, Cherry, JM, Davis, AP, Dolinski, K,
Dwight, SS, Eppig, JT, Harris, MA, Hill, DP,
Issel-Tarver, L, Kasarskis, A, Lewis, S, Matese,
JC, Richarson, JE, Ringwald, M, and Sherlock, G.
(2000) Nat. Genet. 25, 25-29. Hazbun, TR,
Malmström, L, Anderson, S, Graczyk, G, Fox, B,
Riffle, M, Sundin, BA, Aranda, JD, McDonald, WH,
Chiu, C-H, Snydsman, BE, Bradley, P, Muller, EGD,
Fields, S, Baker, D, Yates III, JR, and Davis,
TN. (2003) Mol. Cell 12, 1353-1365. McDonald,
WH, Ohi, R, Miyamoto, DT, Mitchison, TJ,
and Yates III, JR. (2002) Int. J. Mass Spectrom.
219, 245-251.
Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M,
and Seraphin B. (1999) Nature Biotechnol. 17,
1030-1032.