Essential Cell Biology 5th edition

520
HOW WE KNOW
TRACKING PROTEIN AND VESICLE TRANSPORT
Over the years, biologists have taken advantage of
a variety of techniques to untangle the pathways
and mechanisms by which proteins are sorted and
transported into and out of the cell and its resident
organelles. Biochemical, genetic, molecular biological,
and microscopic techniques all provide ways to monitor
how proteins shuttle from one cell compartment to
(A)
radioactively
labeled proteins
+
signal sequence
CENTRIFUGATION
+
+
free protein
protein
co-sediments
with organelle
isolated organelle
PROTEIN TRANSPORT
protein imported
into isolated organelle
PROTEASE ADDED
DETERGENT ADDED
(B)
protease
free protein
degraded
Figure 15−27 Several methods can be used to determine
whether a labeled protein bearing a particular signal
sequence is transported into a preparation of isolated
organelles. (A) The labeled protein with or without a signal
sequence is incubated with the organelles, and the preparation is
centrifuged. Only those labeled proteins that contained a signal
sequence will be transported and therefore will co-fractionate
with the organelle. (B) The labeled proteins are incubated with
the organelle, and ECB5 a protease e15.27-15.27 is added to the preparation. A
transported protein will be selectively protected from digestion
by the organelle membrane; adding a detergent that disrupts
the organelle membrane will eliminate that protection, and the
transported protein will also be degraded.
another. Some can even track the migration of proteins
and transport vesicles in real time in living cells.
In a tube
A protein bearing a signal sequence can be introduced
to a preparation of isolated organelles in a test tube.
This mixture can then be tested to see whether the protein
is taken up by the organelle. The protein is usually
produced in vitro by cell-free translation of a purified
mRNA encoding the polypeptide; in the process, radioactive
amino acids can be used to label the protein so
that it will be easy to isolate and to follow. The labeled
protein is incubated with a selected organelle and its
translocation is monitored by one of several methods
(Figure 15−27).
Ask a yeast
Movement of proteins between different cell compartments
via transport vesicles has been studied
extensively using genetic techniques. Studies of mutant
yeast cells that are defective for secretion at high temperatures
have identified numerous genes involved in
carrying proteins from the ER to the cell surface. Many
of these mutant genes encode temperature-sensitive
proteins (discussed in Chapter 19). These mutant proteins
may function normally at 25°C, but, when the yeast
cells are shifted to 35°C, the proteins are inactivated. As
a result, when researchers raise the temperature, the
various proteins destined for secretion instead accumulate
inappropriately in the ER, Golgi apparatus, or
transport vesicles—depending on the particular mutation
(Figure 15−28).
At the movies
The most commonly used method for tracking a protein
as it moves throughout the cell involves tagging the
polypeptide with a fluorescent protein, such as green
fluorescent protein (GFP). Using the genetic engineering
techniques discussed in Chapter 10, this small protein
can be fused to other cell proteins. Fortunately, for many
proteins studied, the addition of GFP to one or other
end does not perturb the protein’s normal function or
transport. The movement of a GFP-tagged protein can
then be monitored in a living cell with a fluorescence
microscope. In 2008, the Nobel Prize in Chemistry was
awarded to Osamu Shimomura, Martin Chalfie, and
Roger Tsien for the development and refinement of this
technology.