lecture 2 - eukaryotic cells - organisation and key features Flashcards
What is vesicular transport?
the process used to move proteins and lipids between various intracellular compartments
Why is the specificity of vesicular transport important?
An example of why specificity of vesicular transport is important comes from the secretory pathway. Proteins must travel through the secretory pathway in a specified order otherwise they will not receive the correct modifications. Vesicles budding from the ER must fuse with the cis cisternae of the Golgi. If they fused with the plasma membrane they would be secreted before they’ve been properly modified and are unlikely to function correctly (and/or perhaps be secreted at the wrong time). Similarly, if ER-derived vesicles fused with the lysosome then their contents would be inappropriately degraded.
What is the SNARE hypothesis?
two ‘types’ of SNARE proteins; v‐SNAREs on vesicles and t‐SNAREs on target membranes.
each transport vesicle carries a v‐SNARE, which recognizes its partner t‐SNARE on the appropriate target membrane.
v/t-SNARE binding docks the vesicle onto the target membrane and recruits machinery that mediates membrane fusion.
How did Rothman’s experiment measure vesicular transport?
Rothman and colleagues used a biochemical approach to establish a cell free assay to measure vesicular transport. Cell free assay are used by researchers to reconstitute a physiological reaction in a test tube and can be used to dissect cell biological processes.
Rothman’s cell free assay took advantage of the way that glycosylation reactions are compartmentliased in different cisternae of the Golgi apparatus, and followed one protein’s journey through the Golgi. The protein followed was a viral coat protein (from Vesicular stomatitis virus) VSV-G protein . When VSV infects a cell it hijacks the secretory pathway to make components of its capsid – one of these proteins is the G protein which enters the secretory pathway and then travels on through the Golgi receiving glycosylation on the way.
Remember from lecture 1 that glycosylation involves addition of different sugar residues as proteins move from cisterna to cisterna. One of these reactions, that occurs in the medial Golgi, is the addition of a sugar moeity N-Acetyl glucosmaine (GlcNAc) – this reaction is catalysed by GlcNAc transferase, an enzyme found exclusively in the medial Golgi. When VSV-G protein is in the cis Golgi it doesn’t have any GlcNAc residues, but once it is transported to the medial Golgi and encounters GlcNAc transferase it will. It is this transport step that Rothman’s assay follows – from the cis- to the medial-Golgi.
How did Rothman determine that cis-to-medial Golgi transport had occurred?
Key to any cell free assay is the ability to ascertain that the process you are trying to reconstitute has occurred (i.e. a readout that you can follow).
Rothman and colleagues utilsed two tricks to follow cis- to medial-Golgi transport.
- Addition of radioactive GlcNAc to the growth media of cells means that when the VSV-G protein reaches the medial Golgi it gets a radioactive GlcNAc moeity added on to it. Addition of 3H-GlcNAc allows detection of glycoproteins carrying GlcNac
- In mutant cell lines lacking GlcNAc transferase activity VSV-G protein never gets GlcNAc moeities added to it (even when it is in the medial Golgi because the enzyme isn’t there). Mutation of GlcNAc Transferase prevents addition of GlcNAc to glycoproteins
How did Rothman utilise two cell lines?
The assay used two different cell lines (both are Chinese hamster ovary - CHO - cells which are very commonly used in cell biology as they’re relatively easy to work with.
One of the cell lines had been infected with VSV and therefore had VSV-G protein travelling through its secretory pathway, but this cell line is missing GlcNAc transferase and so that protein doesn’t get GlcNac added to it.
The other cell line is an uninfected wild-type (it has GlcNAc transferase) been grown in the presence of radioactive GlcNAc (note that this cell line has no VSV-G protein as it hasn’t been infected).
How was the assay carried out in Rothman’s experiment?
To carry out the assay, the two different cell lines were lysed and then their internal organelles mixed together in a test tube.
Appearance of radiolabelled VSV-G protein gives a measure of cis- to medial-Golgi transport because the only way that VSV-G protein could become labelled with radioacitve GlcNAc in this system is if it’s been transported from the cis- cisternae of the donor cell line to the medial of the acceptor.
Only G-protein transported from donor (cis) to acceptor (medial) will incorporate 3H-GlcNAc
What is required to reconsitute cis-to-medial Golgi transport?
In order to reconstitute cis- to medial-Golgi transport organelles from both cell lines are required (donor and acceptor; as source of the VSV-G protein and GlcNac Transferase respectively), as well as addition of energy in the form of ATP and cytosol. This demonstrated that there is something in cytosol that’s required for the transport to occur.
It was discovered that if cytosol had been treated with a sulphydryl alkylating agent called NEM it couldn’t support the assay. Demonstrating that there is a factor in the cytosol, required for the assay to work, that is senstive to NEM - named NSF (NEM-sesnitive factor). It was then discovered that addition of fresh cytosol could overcome this inhibition.
Successive fractionation of cytosol identified NSF as a 76kD polypeptide (i.e. addition of this single protein to NSF-treated cytosol resotres its ability to support cis- to medial-Golgi transport). This was first identification of a molecule required to transport from one membrane-bound organelle to another.
Given that NSF is found in cytosol, it was reasoned that to carry out a role in vesicular transport and membrane fusion NSF must bind to something in the membranes themselves. Subsequent experiments used NSF to identify SNARE proteins as being the factors that bind NSF to membranes.
What are SNAREs?
SNAREs are a large protein family. Individual cells have many different types of SNARE proteins localised to different organelles. This led to formulation of the SNARE hypothesis.
Each membrane transport step requires specific factors (e.g. SNARE proteins) as well as more general machinery (e.g. NSF).
SNAREs = protein superfamily (>60 in mammalian cells)
Different members localise to different membranes
Why did Schekman use yeast in his experiment?
Schekman and colleagues used a different approach to identify membrane traffic machinery. Yeast genetics to identify mutants defective in secretion
Used the yeast S. cerevisiae, a commonly used model eukaryote. Has a secretory pathway analogous to that of mammalian cells. Proteins travel through the ER, through the Golgi and are then packaged into secretory vesicles that fuse with the plasma membrane.
As well as being used to secrete proteins from the cell, these secretory vesicles are important for cell division. When the vesicles fuse with the plasma membrane, this increases the surface area of the cell, allowing a bud to form. Once this reaches a certain size (following fusion of more secretory vesicles with the plasma membrane) it forms a separate daughter cell.
Why did Schekman use a genetic screen?
Schekman and colleagues reasoned that mutants defective in secretion would be more dense than wild-type cells
Genetic screen are often used to identify machinery required for a process by generating mutants that are defective in the process of interest and then determining which gene(s) are mutated.
Key to a good genetic screen is the identification of an appropriate phenotype to select for.
To identify mutants defective in secretion cells that were more dense than normal were selected (the rationale being if the secretory isn’t functioning the cells won’t divide, but will become more dense as membrane will still be produced and build up inside the cell).
Because secretion is an essential process in yeast (required for cell division) the screen set out to identify conditional mutants (in this case temperature sensitive). Looking for mutations that would let the cells secrete (and grow) normally at 25°C but couldn’t at elevated temperatures (and therefore become more dense).
How did Schekman use a genetic screen?
To carry out the screen a population of yeast cells were subject to mutagenesis (e.g. UV light) – introduces random mutations into the genome. These cells were then incubated at elevated temperatures to allow any cells that had become defective in secretion as a result of this mutagenesis to become more dense. The cells were then loaded on top of a density gradient – a matrix that separates on basis of density – two discrete populations were see, one more dense than the other (note that control/unmutated cells are all the same density – that equivalent to the less dense population of the mutated cells).
sec mutants isolated by piercing the tube with a syringe and extracting the cells.
These could then be plated out (at 25°C) and individual colonies isolated (each colony coming from a single cell).
To identify which gene was mutated and causing the secretion defect individual mutants were transformed with a genomic library prepared from wild-type cells.
This library consists of individual ORFs from the yeast genome expressed from plasmids. Each yeast cell will take up one plasmid – those that take up a wild-type copy of the gene whose mutation causes the secretion/growth phenotype will now be able to grow at 37°C (restrictive conditions).
The plasmid rescuing that mutant can be isolated from the cells and sequenced to reveal the identity of the gene.
What were the results of Schekman’s genetic screen?
The original sec screen identified 23 different gene products as being required for secretion.
These were categorised according to the stage of the secretory pathway they blocked (determined by electron microcopy) e.g. sec12 mutants accumulate ER, sec7 mutants accumulate Golgi, sec1 mutants accumulate secretory vesicles.
Sequencing of SEC18 revealed high homology to NSF and subsequent experiments demonstrated that the Sec18 and NSF are functionally equivalent (expression of human NSF rescues the secretion phenotype of yeast sec18 mutant cells), demonstrating that molecular mechanisms controlling membrane traffic are conserved through eukaryotic evolution from yeast to humans.
