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