Biomedical Engineering Reference
In-Depth Information
Chapter 6
Investigating Signaling Processes in Membrane Traffi cking
Laura J. Sharpe and Andrew J. Brown
Abstract
Signaling and phosphorylation can be very diffi cult areas to explore, as there can be a lot of cross-talk
between signaling pathways, and the stoichiometry of phosphorylation is often very low, and is typically
transient. Here we describe an innovative assay using an immunoprecipitation approach, followed by a
kinase assay, coupled with a phosphorylated substrate-specifi c antibody. We also indicate a database and
prediction program that can be used in these situations.
We apply these methods to investigate the regulation of ER-to-Golgi traffi cking by protein phos-
phorylation of critical components in the traffi cking machinery. Key components of this transport step are
well known thanks to the pioneering work of the 2013 Nobel Prize winners James Rothman, Randy
Schekman, and Thomas Südhof. However, the regulation aspect of this process is relatively unexplored.
Key words
Akt, Immunoprecipitation, Sec24, Kinase, Phosphorylation
1
Introduction
The vesicle transport system within cells has been largely elucidated
by the 2013 Nobel Prize winners Randy Schekman, James
Rothman, and Thomas Südhof. Firstly, Schekman uncovered the
genetic basis for the secretory pathway components [
1
,
2
], followed
by Rothman discovering the proteins involved in vesicle fusion
with membranes [
3
]. Südhof then determined how vesicles know
when to release their contents [
4
].
The fi rst part of the secretory pathway involves the transport of
proteins from the endoplasmic reticulum (ER), where they are syn-
thesized, to the Golgi, where they are sorted and distributed [
5
].
This process involves the COPII complex, which consists of a
large number of subunits. These were identifi ed from the landmark
experiments by Randy Schekman and Peter Novick. These investi-
gations hypothesized that yeast cells with a mutation in any of the
genes that encode components of the secretory pathway would be
lethal. This led to screening of temperature-sensitive mutants, and
ultimately, many of the “Sec” proteins were identifi ed in yeast [
1
].
