A road map for cell biology: Why studying large protein

complexes is crucial at this time

David Drubin, UC Berkeley

Why study large complexes?

• Proteins typically function in association with other proteins.

• Protein complexes are important for virtually every biological process and most diseases.

• Genome sequences identify tens of thousands of genes: linking these to 200-300 core biological processes will make their study manageable.

• Recently developed and/or improved technologies and methodologies make studies of large complexes more feasible and informative.

A brief tour of some protein complexes and their biological

and biomedical importance

(from the Alberts et al. textbook)

Nucleosome

DNA Polymerase

Nuclear pores

Ribosome

Proteosome

Cell Cycle: Short-livedcomplexes

COPII-coated vesicle

MT motor - vesicle

Enzyme (Aspartate transcarbamoylase); two conformations

Red blood cell skeleton

Signal transduction complex

Many new and improved approaches for studies

of protein complexes are now available

Using combinations of these approaches makes

the most of their complementary features

These approaches can be used in targeted or genome-wide studies

Approaches to studies of protein complexes

Structural Proteomics

Biological function mechanism regulation

Proteinlocalization

Biochemicalactivities

Proteinstructure

Expressionstudies

Protein-proteininteractions

Geneticinteractions

Mutant/RNAi/Chemicalphenotypes

Post-translationalmodifications

Features of genome-wide,non-targeted “big science”

*Efficiency

*Unbiased

*Patterns can emerge that only are evident when analyzing large datasets

*Validation and follow-up, quality control

Pros:

Cons:

Features of targeted studies “small science”

*Science is an iterative process -much can be missed with one pass

*Appreciation of vital subtleties of a system, knowing where to look and what to look for

Cons:*Less efficient

Pros:

*Tools and insights for validation and quality control

Non-Targeted“Big Science”

Targeted“small science”

Technologists

Biologists

Mass spectrometry is having a revolutionaryeffect on identification of subunits of protein complexes and their post-translationalmodifications

Mass Spectrometry

The Budding Yeast Kinetochore

+ TEV Protease

ZZSKinetochore ProteinIgG

Sepharose

S ProteinAgarose

InteractingProteins

SKinetochore ProteinInteractingProteins

Mass Spectrometry

Elute withUrea

(Scott Andersonand John Yates)

Molecular Analysis of KinetochoreComposition and Organization

3 novel kinetochore sub-complexes defined

28 kinetochoreproteins purified

(75% of total)

5 novel kinetochore proteins identified

Light microscopy can verify the in vivo relevanceof proteomically observed associations, and provide valuable information about dynamics

Light microscopy

Dad3p and Dad4p Localize toSpindles and Kinetochores

Spindle(Intact Cells)

Kinetochore(Chromosome Spreads)

Tandem Mass SpectrometryMapping of Phosphorylation Sites

Dam1p Complex -

Ndc80p Complex -

Ctf19p Complex -

Ipl1p Complex -

in vivoPhosphorylation Sites

13

1

4

0

Ipl1p Targets

6

1

3

0

Genetics can verify in vivo relevance of post-translational modifications, associations, etc.

Genetics

dam1 (S to D) MutantsSuppress ipl1-2

dam1 (S to D)

ipl1-2 dam1 (S to D)

Wild Type

ipl1-2

The current demand for mass spectrometry outstrips the

existing capacity

• Cell cycle stages

• Response to stimuli and perturbation

• Affects of various mutants

Clone John Yates

Two hybrid Arrays

• Yeast Resource Center (Stan Fields)

• All yeast ORFs on 16 plates

HIGH QUALITY datasets from large-scale, genome-wide analyses such as two-hybrid screens,mass spec of complexes, etc., can greatly accelerate biological research.

Datasets from non-targeted proteomics

YeastWASp

GFP, its spectral variants, and DsRed are drivingthe need for advanced imaging systems tostudy protein complexes in vivo

-High speed multi-color analysis-FRET-FRAP-Automated image acquisition and analysis

Light microscopy is critical fordetermining where and when proteins associate in complexes

Endocytic protein interactions

Why doesn’t the whole cytoplasm precipitate?

Interactions are regulated in space and time!

Small molecule inhibitors can be powerful toolsto switch on and off associations within proteincomplexes in vivo

Chemical Biology (Genetics)

Summary

• Protein complexes are involved in virtually every cellular process and disease

• Complete genome sequences combined with availability of new technologies make studies of protein complexes crucial at this time

• The focus of all such studies should be on illuminating biological and disease mechanisms

Acknowledgements

• Yeast Resource Center (John Yates, Stan Fields, Trisha Davis)

• Kevan Shokat (UCSF)• Barnes and Drubin labs (UCB)