Cell Biology Lecture No. 4: Protein Synthesis & Transport
Monday January 21 , 2013
LECTURE 3 CONT’D
Detecting Specific Proteins:
-Western blotting (or immunoblotting) is a process used to detect specific proteins. First, an
electric current is used to transfer the proteins on the SDS-polyacrylamide gel onto a
membrane. This membrane is then incubated with primary antibodies that recognize your
protein of interest. Enzyme-labelled secondary antibodies are then linked to the bound primary
antibodies. Finally, a substrate is added to the membrane that is catalyzed by the enzyme and
gives off luminescence (enhanced chemiluminescence), showing you where your protein of
interest is located. This last step is very rarely used and more often fluorophores are attached to
the secondary antibody. The luminescence makes an impression on the X-ray film and you can
see darker banding patterns indicating higher expression.
Protein Sorting & Protein Targeting:
-In a typical mammalian cell, tens of thousands of different proteins are made and it becomes a
major issue for the cell to direct them to the right destination. Targeting involves signal
sequences directing proteins to their appropriate destinations (organelles) during or after
synthesis. Sorting involves the directing of proteins involved in secretory pathways (that make it
into the ER, Golgi body and lysosomes).
General Principles & Overview Of Protein Synthesis, Sorting & Targeting:
-Many proteins are synthesized by free-floating ribosomes in the cytosol that can be broken up
into two categories: Proteins which function in the cytosol and proteins which are targeted (by a
specific signal sequence) to function in intracellular organelles like the ER, mitochondria,
chloroplasts or the nucleus. Other proteins are synthesized by ribosomes bound to the Rough
Endoplasmic Reticulum and can also be broken up into two categories: Proteins which function
in the ER and proteins which are sorted to function near the plasma membrane, Golgi body or
lysosome. Accordingly, we see that two pathways arise for proteins: nonsecretory proteins and
-In the cytosol, mRNA transcript is recognized by the translational machinery and synthesized
into protein. Once fully synthesized, some proteins are tagged with specific signal sequences
which target the protein to mitochondria, chloroplasts, peroxisomes, or the nucleus. Other
proteins have a signal sequence which targets the nascent polypeptide (as well as the
associated ribosome) to the RER, where the signal sequence binds to a receptor and translation
is completed in the lumen. Translocation channels (pores) help secretory proteins cross the
pesky hydrophobic membrane (which takes energy to do).
The Structure Of The Endoplasmic Reticulum: -The first part of the Endoplasmic Reticulum is actually a direct continuation of the nuclear
membrane, though they do become a collection of stacked tubules (cisterna) that are distinct
from the nuclear membrane. The association of ribosomes with its membrane is what defines
Studies On The Secret Lives Of Secretory Proteins:
-A radio-labelling experiment was performed to answer how certain proteins are secreted. This
included using amino acids with a radioactive signature that are taken up by a cell culture and
incorporated in the building of proteins. After homogenization, it was discovered that open
membranes of RER fragments have a tendency to re-form and become microsomes. Sucrose
density-gradient centrifugation was then used to concentrate the microsomes (RER matter).
Half of this centrifuge was treated with detergent (to break apart microsomal membrane) and
protease (to break apart proteins inside) and half was treated with just protease. The conclusion
of this experiment is that secretory proteins need to enter the RER, where proteins are either
made or imported.
Functions Of The Rough Endoplasmic Reticulum:
-Secreted and membrane-bound proteins are sorted through the RER. Post-translational
modification such as the adding of sugars and carbohydrates to polypeptides is performed in the
RER. Other structural features such as disulphide bonds are added in the RER as well.
Chaperones assist newly translated polypeptides to fold correctly in the RER.
The Simultaneous Occurrence Of Translation & Translocation:
-Another experiment was performed to determine if proteins are made, then imported into the
RER or if they are made and imported simultaneously. In a cell-free system (in vitro protein
translation system), the mRNA of a known secretory protein is translated. When these
completed proteins with their signal sequences were introduced to microsome membranes, they
wouldn’t enter. The cell-free experiment demonstrated that translocation of secretory proteins
into microsomes is coupled to translation.
Major Players In The Rough Endoplasmic Reticulum:
-The major players associated with the RER include: the amino terminal signal