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The Yeast
Resource Center
An NCRR Biomedical
Technology Resource Center
http://depts.washington.edu/~yeastrc
Focus
• Technologies that extract information
about macromolecular interactions
from the genome sequence.
• Regulation
• Dynamics
The people
The technologies
• The YRC is a consortium of five
groups and five technologies
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–
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–
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Aebersold
mass spectrometry
Yates
mass spectrometry
Fields
two-hybrid
Baker
protein struct. prediction
Muller/Davis microscopy and FRET
Davis/Baker informatics
A P41 center has
three missions
• Technology development
• Collaboration
• Dissemination and Training
By the numbers
(for 2002)
• 51 publications
• Technology development:
– 19 projects
• Collaborations:
– 132 collaborators, 80 projects
• Dissemination and Training:
– 130 individuals
• 26 states, 12 countries
Unknown ORFs
• 105 unknown essential ORFs
• Integrate all our technologies
• Find a balance between quantity of
data and quality of data
Analysis of high throughput data
von Mering et al., 2002
Unknown
essential ORFS
• Tag with TAP tag and YFP tag
• Purify each with TAP tag
• Identify copurifying proteins by
mass spec
• Localize in living cells
• Two-hybrid analysis
• Advanced homology searches
• Protein structure prediction
Optimization of purification
with TAP tag
212
212
97
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66
56
43
36
Graczyk, Sundin, Fox and Davis
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97
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66
56
*
*
43
*
36
*
*
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*
Summary of
purification
• 20 are part of a complex
– 15 different complexes
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•
•
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28 purify by themselves
22 could not be tagged
26 were inactivated by tag
9 were not produced at detectable
levels in logarithmic cultures
Integration of technologies
builds the story
• New complex, ribosomal recycling
– All four members are essential
unknowns
– One localizes to nucleolus, three
others killed by tag
– Two-hybrid confirms mass spec
– Homology searching finds homology
of one to ribosomal recycling factor
Integration of technologies
builds the story
• An ER protein that purifies alone
– Mass spec finds only that protein
– Localizes to ER
– Two-hybrid identifies a collection of
membrane proteins
– Homology searching identifies
homology to mannosyltransferases
Integration of technologies
builds the story
• YKR079c-TAP copurifies with 2 proteins. But
localization reveals they are likely to form two
complexes of two proteins.
YKR079c
NUC1
YMR099c
Lessons
• Mass spec is extremely powerful
• Two-hybrid expands the story and
provides critical information for proteins
not in complexes
• Localization is fast and easy
• Great deal of information to be had
merely by advanced homology searching
• Protein structure prediction is underway
Recommendations
• Think in terms of optimal throughput which
strikes a balance between quantity and quality.
• More mass spec capacity is needed and the
technology is not yet readily disseminated.
• Promote the integration of technologies.
• Increase capacity and speed of advanced
homology searches.
Yeast Resource Center
• John Yates
– Scott Anderson
– Hayes McDonald
• Ruedi Aebersold
– Rick Newitt
– Eugene Yi
• Stan Fields
– Tony Hazbun
– Jennifer Aranda
• Eric Muller
– Brian Snydsman
• Trisha Davis
– Bethany Fox
– Beth Graczyk
• David Baker
– Lars Malmström
– Bill Schief
• Informatics
– Mike Riffle