Cell News | Issue 01, 2017 - page 20

Cell News 01/2017
Quantitative mapping of protein
subcellular localization changes
Daniel N. Itzhak, Stefka Tyanova, Jürgen Cox, and Georg H.H. Borner
Presenting author: Georg H. H. Borner
Max Planck Institute of Biochemistry, Department of Proteom-
ics and Signal Transduction, Am Klopferspitz 18, 82152 Martin-
sried, Germany
Subcellular localization critically determines protein function,
and many biological processes involve changes in protein
localization. We have developed a method for the global cap-
ture of protein translocation events. The approach combines
subcellular fractionation with quantitative mass spectrometry,
to determine protein localizations on a proteome-wide scale.
We first applied the approach to generate a comprehensive
localization map from HeLa cells, covering 8,700 proteins. All
major organelles were resolved, and protein localizations were
assigned with very high accuracy (estimated at >92%). The
method also provides absolute protein abundance information
(protein copy numbers per cell and compartment), allowing us
to construct a quantitative model of HeLa cell anatomy. Impor-
tantly, organellar maps are highly reproducible, and can hence
be used comparatively. To demonstrate this, we applied maps
to investigate protein dynamics during EGF signaling. Numer-
ous known and novel EGF-induced protein translocation events
were captured, such as EGFR endocytosis, adaptor recruit-
ment, and nuclear transitions. Combining the translocation
data with protein abundance estimates allowed us to derive a
first quantitative model of EGF-triggered protein movements.
Organellar maps offer an unbiased systems-level analysis tool
for the quantitative characterization of cellular processes. The
approach does not require any process-specific reagents, and is
hence applicable within a wide range of biological contexts.
Itzhak DN, Tyanova S, Cox J, Borner GH (2016) Global, quanti-
tative and dynamic mapping of protein subcellular localization.
Elife, e16950.
Purification & characterization of Plasmodium tubulin –
A novel lead to anti-malarial drug discovery
William Hirst, Dr. Simone Reber
Presenting author: William Hirst
IRI für Lebenswissenschaften, Humboldt-Universität zu Berlin
Microtubules are long, hollow filaments formed by the polym-
erization of
tubulin heterodimers. In eukaryotic cells, they
facilitate intracellular transport and are essential components
of organelles such as flagella and the mitotic spindle. Drugs
that interfere with microtubule function are used to treat a
broad range of maladies, including malaria. However, the pu-
rification of assembly-competent tubulin is not possible using
recombinant expression systems, which has until recently pre-
cluded direct in vitro studies of drug interactions with tubulin
from diverse organisms such as Plasmodium falciparum, the
parasite that causes the most deadly form of malaria.
We have recently taken advantage of the high binding affinity
of tubulin to the Tumor-Overexpressed-Gene (TOG) domains
of Stu2, a yeast microtubule polymerase, to efficiently isolate
active tubulin from multiple eukaryotic cell types via affin-
ity chromatography, thus eliminating a major limitation in
microtubule research. Following this approach, we have for the
first time purified native tubulin from Plasmodium falciparum.
Importantly, the protein is free of host tubulin contamination,
allowing for an accurate representation of parasitic microtu-
bule behavior. We have developed a framework for investigat-
ing tubulin as a novel drug target and are now able to charac-
terize microtubule dynamics in a new biological context.
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