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– 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 von Mering et alet al., 2002., 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
*
*
*
*
212
43
56
97
66
36
Standar
ds
YGR046W
(44k
D)
YFR003C
(18k
D)
YGR002C
(55k
D)
YJR01
3W (3
5kD)
YKR079C
(97k
D)
Spc97p
*
*
*
*
*
*
212
43
56
97
66
36YHR08
5W (3
8kD)
YKR038C
(47
kD)
Standar
ds
YGR024C
(28k
D)
YNL158W
(22k
D)
Spc97p
YOL03
4w (1
26 k
D)
Optimization of purification with TAP tag
Old
met
hod
Graczyk, Sundin, Fox and Davis
New
met
hod
Summary of purification
• 20 are part of a complex – 15 different complexes
• 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
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