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Lecture 10

BIO315H5 Lecture 10: BIO315 – Lecture 10

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Hai- Ying Mary Cheng

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BIO315 Lecture 10
Lecture 10
Question From Lecture 9:
What are the 2 major factors that provide directionality to nuclear transport processes?
Concentration gradient b/w Ran-GTP and Ran-GDP
Nuclear import and export receptors that bind to Ran behave very differently
Import receptors: release and dissociation of cargo - when Ran binds, the receptor
undergoes a conformational change that does not allow it to bind to the cargo
Export receptors: when the receptor binds to Ran it undergoes conformational change
that allows it to associate with the cargo (reverse of import)
ER Trafficking
The Endoplasmic Reticulum
Very large organelle in the cell
Makes up 1/2 of the total membrane of a typical animal cell
Made up of a network of tubules and flattened discs
ER membrane is continuous with nuclear envelopes
Inside of ER is called the lumen
Major function of the ER are for synthesis of proteins and lipids and for storage of
Image is an animal cell expressing an ER membrane protein fused with GFP
2 major functions:
1. Protein and lipid biosynthesis
All proteins that end up in the lumen of the ER are all made in the ER
Transmembrane proteins are synthesized on the ER membrane
1. Serves as a major intracellular store of Ca2+
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Structural and functional diversity of ER: rough vs. smooth ER
Rough ER - looks rough b/c of ribosomes on surface of ER - on the cytoplasmic leaflet of
the ER membrane
As proteins are synthesized on the membrane they can be translocated to the lumen
There is functional specialization in distinct regions of the ER to serve distinct needs of
the cell
Rough ER is coated with ribosomes on the cytosolic face
Protein synthesis occurs on the surface of the ER
Protein is translocated to the lumen of the ER as it is being made
In other organelles such as mitochondria, nucleus and peroxisomes, translocation occurs
Functions of smooth ER
Site where you have production of lipids
Also site of production of lipoprotein particles
Has enzymes important for detoxification
Storage of Ca2+ ions
You need Ca2+ for signalling processes and for muscle cell contraction
Some cell types have a greater abundance of smooth ER. E.g. Cells that synthesize
cholesterol have an abundance of smooth ER.
For example, liver cells (called hepatocytes) also have an abundance of smooth ER. The
ER houses many different enzymes needed to detoxify lipid-soluble drugs and other
harmful metabolic by-products (cytochrome P450 family of enzymes).
For calcium storage, ER contains a high concentration of calcium-binding proteins. In
muscle cells, release of calcium causes muscle contraction and reuptake causes
Co-translational vs. post-translational
When proteins are synthesized on the surface of the rough ER they are translocated into
the ER - this type of translocation occurs co-translationally meaning that the
translocation/movement into the ER happens as the mRNA transcript is being translated
into a protein
Co-translationally: Movement of protein into the ER (translocation) happens as mRNA
transcript is being translated into a protein
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However, there are cases where the protein is translocated post-translationally meaning
after the protein is fully synthesized
Purified microsomes to study ER function
You can isolate the membrane of the ER by using microsomes
Mechanical force is used to break up the membranes of the cell
When you break up membrane structures, pieces of the ER will spontaneously form
closed vesicles called microsomes
These microsomes can then be purified by sucrose gradient centrifugation - process:
take preparation and layer it on top of a tube that has a sucrose concentrated solution
and you spin it to set up a sucrose gradient where the different cellular components are
going to end up as a gradient
Hydrophobic tails are buried inside with hydrophilic head groups facing outwards. This is
energetically most favourable - sucrose gradient centrifugation
Microsomes are going to end up in specific positions based on their density
Rough microsomes are derived entirely from rough ER
In contrast, smooth microsomes can come from smooth ER, but also microsomes
derived from the Golgi, endosomes, or fragments of the plasma membrane
An ER signal sequence directs a growing polypeptide chain to the ER
There are 2 broad categories of proteins that are translocated to the ER
1: The first are water soluble proteins that reside in the lumen of the ER. These are
water-soluble, and the entire protein is translocated into the ER
2: The second are proteins that are expressed on the ER membrane or on cell
membranes derived from the ER. These include the plasma membrane, Golgi, etc. -
they get trafficked to the lumen of the Golgi or other vesicles
These transmembrane proteins are only partly translocated
Both types of proteins have an ER signal sequence that initiates the translocation, which
occurs by a common mechanism
The Signal Hypothesis
States that in order for proteins to be directed to the ER and translocated to the ER
there needs to be a signal that brings them there - this signal ER sequence
Soon after the translation of the N-terminal leader peptide and its emergence from the
ribosome, this sequence is recognized by a translocator complex on the ER membrane
Translocation occurs as the translation continues
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