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First Class Lecture notes Dynamic Cell Module
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- Dynamic Cell Module
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- University Of Bath (UoB)
Dynamic cell lectures 2-4
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PW DYNAMIC CELL LEC 2-4SACCHAROMYCES CEREVISAE
• Schekman et al. carried out research using S. cerevisae aiming
to identify which genes/ proteins were involved in protein
secretion. These experiments had to be carried out on a simple
organism, and yeast have similar organelles to animal cells (as
both eukaryotic organisms) so S.cerevisiae was ideal.
• They made the assumption that total loss of function of
proteins required for secretion would be lethal to yeast, as
protein secretion is an essential function of the cell
• If you chemically mutate a population of yeast by exposing
the cells to a chemical mutagen, they will individually pick up
different mutations (of unknown localisations). Some of these
mutations will be lethal in which case the cells will die and not
be part of the population, so will not form colonies.
• Schekman took the approach of looking for those (mutated cells) cells that could
form colonies (grow) at a permissive temperature (25 C) but could not grow at 37 C –
conditionally lethal mutants (temperature sensitive mutants). You can do this by a
technique called replica plating: one plate of yeast cells without lethal mutations
grown at the permissive temp of 25 C, the other at a higher temp of 37 C. Wild-type
yeast cells would grow at both temperatures, whereas those with ts mutations would
not grow at the higher temperature.
• They then screened the yeast that were conditionally lethal (temperature sensitive, ie
could not grow at 37 C) for those that had defects in secretion (could secrete
proteins at 25 C but not at 37 C). One of the proteins studied was the invertase
enzyme, which converts sucrose (disaccharide) to glucose and fructose
(monosaccharides).
• Why did the ts mutants fail to grow at 37 C? These colonies have a mutation which
causes an altered amino acid sequence and protein which cannot fold correctly to
perform its function at higher temperatures (destabilised). This may impair protein
secretion from the cell.
• SEC mutants show an increase in buoyant density during a period of 1−3 hours
after a shift to the restrictive growth temperature (37 °C). They found that the first
mutant they isolated was denser at high temp than low temp (probably due to
accumulation of secretory organelles). Used this finding as a basis to enrich for
further secretory mutants-identified a further 23.
,SEC1 MUTANT
• Cloning of SEC genes/ identification of proteins
involved in secretion: first secretory mutant isolated was the
SEC1 mutant. At a growth temperature of 25 C the yeast look
morphologically normal and can grow. When shifted to 37 C
the yeast accumulate vesicles (and can no longer
grow/multiply). Hypothesised that mutations in SEC1 may
cause defects in diffusion across the plasma membrane,
leading to a build-up of secretory vesicles within the cell. Hence, yeast cell cannot
secrete proteins at this high temperature.
• How do you identify where the mutation is (which gene is mutated) and hence
the defective protein? Complementation – can make a genomic library of yeast
plasmids with different gene inserts. Transform library into mutant yeast cells, then
cells grown at 37 C, and cells that have taken up plasmid with insert have
complemented mutation so can grow at 37 C. The inserted gene sequence binds to
its complementary sequence (mutated gene) in the yeast DNA, preventing it from
producing the defective protein. Then isolate plasmid from these cells and the
sequence insert of the plasmid. Can therefore identify the DNA sequence that has
complemented the defect, and translate this into a protein, identifying the protein
responsible for the defect in the protein secretory pathway. These experiments
allowed discovery of molecules involved in protein secretion.
• Full method: Isolation of Secretory (sec) Mutants. X2180-1A cells were grown in YPD
medium and treated with 3% ethyl methanesulfonate for 60 min at 25°C; the survival
rate was 50-70%. The mutagenized culture was diluted with an equal volume of 12%
sodium thiosulfate, and the cells were centrifuged and washed twice with distilled
water. The cells were then grown in YPD medium for 8 hr at 240C, and diluted
aliquots were spread on minimal medium agar plates. After 3 days at 22°-24°C, 1600
colonies were replica-plated onto YPD medium and incubated overnight at 370C. The
temperature-sensitive clones (87/1600) were replica-plated onto phosphate-free
minimal medium to derepress the synthesis of acid phosphatase, and after 10 hr at
240 or 370C the replicas were stained for secreted acid phosphatase (8). The clones
that showed temperature-sensitive secretion of phosphatase were screened for
conditional secretion of invertase. Cultures grown at 24°C in liquid minimal medium
containing 5% glucose were shifted to fresh medium containing 2% sucrose, and
after 5 hr at 240 or 370C the cells were centrifuged, washed with distilled water, and
assayed for secreted invertase. Two clones showed conditional secretion but normal
incorporation of 35SO42- into protein at 370C (data not shown). The mutant loci
designated sec 1-1 and sec 2-1 are nonallelic and recessive. Only sec 1-1 will be
described in this report.
YEAST SEC MUTANTS
, • Mutants were classified according
to their morphology when grown
at 37 C: for example, the SEC1
mutant was divided into Class E,
characterised by accumulation of
protein into secretory vesicles. At
this high temperature, other Sec
mutants display a variety of other
defects in the protein secretory
pathway – in Class B proteins accumulate in the ER, and in Class D they accumulate in
the Golgi apparatus.
• Double mutants provided further evidence of the order of events in the secretory
pathway… for example, if Class B mutant crossed with Class E, this would produce
yeast with both mutations. Upon shifting these cells to a higher temperature, would
expect to accumulate proteins in ER organelles as these are first in the secretory
pathway (would not accumulate secretory vesicles as proteins would never reach the
vesicles).
• Can also identify post-translational modifications that occur to proteins in different
organelles of the secretory pathway (glycosylation, cleavage, etc.). Secreted protein
expressed in a Class B SEC mutant and cell shifted to
37 C, meaning that the protein would accumulate in
ER, allowing identification of any modifications that
have occurred up to this point. Can then repeat this
experiment on Class D mutants to identify any further
modifications once the protein has reached the Golgi
apparatus.
ORGANELLES OF THE ENDOMEMBRANE
SYSTEM
• ER
• Golgi
• Lysosome
• Endosomes
• These organelles have distinct protein/lipid compositions and carry out different
functions. Do not think of these organelles as static structures.
THINGS TO CONSIDER
• How do molecules (proteins, lipids) move between compartments – what are the
mechanisms?
, • How are compartments created and maintained? E.g. Golgi apparatus has multiple
stacks, each with its own set of processes. How do the enzymes that carry out these
processes get localised to specific Golgi stacks? Once the compartment is created,
how is the composition maintained whilst there is continuous trafficking in and out of
these organelles?
• Specificity: organelles have different compositions (protein/lipid contents). Requires
molecules to be selectively trafficked to certain organelles.
• Composition is related to function. What is the function?
MODIFICATION OF PROTEINS IN THE ER
• ER is the first stage where all proteins in the endomembrane system are assembled –
either integral membrane proteins into the ER membrane, or lumenal proteins
transported into the organelles in the endomembrane system.
• Addition and processing of carbohydrates - Proteins that have the sequence
NXS/T present in the lumen of the ER are often glycosylated (N-linked glycosylation).
A core oligosaccharide is transferred from dolichol phosphate (a lipid) onto the
acceptor site (NXS/T) of the protein. The transfer is catalysed by an ER resident
protein complex called oligosaccharyl transferase. The oligosaccharide is trimmed
and modified further in the ER. This process of trimming and modification plays a role
in protein folding. Most proteins that enter endomembrane system do get
glycosylated, unlike proteins in cytoplasm of cells (active site of oligoscaccharyl
tranferase is in lumen of ER).
• Disulphide bond formation - cytoplasm is reducing environment so proteins here
do not have disulphide bonds, but proteins in the endomembrane system do. E.g.
immunoglobulins. Both intermolecular and intramolecular disulphide bonds can be
formed.
• Assembly into multimeric proteins – e.g. immunoglobulin, which consists of two
heavy chains and two light chains, held together by disulphide bonds.
• Protein folding/quality control - takes place in the ER. Proteins that are misfolded
tend to stay in ER as they associate with chaperones which prevent them from leaving
ER. Many different classes of chaperone proteins (such as BiP, calnexin, Calreticulin).
Only when protein is folded correctly so chaperone cannot bind and is released can
protein leave ER.
• GPI anchors can be added - type of lipid modification. GPI anchors anchor proteins
into the luminal leaflet of membrane, if this protein reaches cell surface it can be
anchored to PM not by transmembrane domain but by GPI anchor e.g. prion protein
is an example of a GPI anchor protein
• Cleavage of signal sequences
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