Tutorial 7 – Secretory Pathway

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Unit 7
Endomembranes
Unit 7
SECRETORY PATHWAY:
Secretory Pathway
• Proteins are synthesized on the Rough ER.
• Move via vesicles to Golgi
• Move via vesicles to Plasma membrane
- Budding is through
coated vesicles (ie.
clathrin)
- Docking is through
the SNAREs
Budding via Coated Vesicle
- Clathrin coated vesicles originate from Golgi
and Plasma membrane
Docking via SNARES
Vesicles are targeted by
interactions between vSNARES and t-SNARES
• each vesicle has unique
v-SNARES embedded in
its membrane
• each target membrane
has unique t-SNARES (vSNARE receptors)
embedded in the target
membranes
Protein Transport: 3 mechansims
1. Nuclear import
2. Protein translocators - protein pores in
the membrane that help unfolded proteins
get into membranes
3. Transport vesicles - membrane vesicles
pinch off a donor compartment, fuse with a
recipient compartment and carry both:
- soluble proteins in lumen and
- membrane proteins associated with
bilayer
Protein Targeting
•
Sorting signals are necessary to direct a
protein to a particular organelle.
1.
2.
3.
4.
Nuclear localization signal
ER signal sequence
KDEL - retention in the ER lumen
Mitochondria/chloroplast signal sequence
NOTE: Proteins destined for nucleus, chloroplast,
mitochondria, and peroxisome are made in cytosol on
free ribosomes!
Nuclear localization signal
- On soluble, folded nuclear proteins, made
on free ribosomes in the cytosol
- Contains one or two short sequences with
positively charged amino acids
- Bind Nuclear import receptors in cytoplasm
- Move into nucleus through nuclear pores
ER Signal Sequence
- Protein Translocator = at N terminal
- Consists of hydrophobic amino acids.
- Causes proteins meant for secretory
pathway to enter the ER
- Retention signal = KDEL
- Used to target “resident proteins” of ER
to get back to ER from Golgi after
sorting
Mitochondria/chloroplast
Signal Sequence
-
-
Protein translocator for proteins made on
free ribosomes destined for mitochondria
and chloroplast
signal sequence at N terminal
What if the sequence is moved to C terminal?
Unit 7
Models of Golgi Function
There are two competing theories:
Cisternal progression model:
New cisternae form continuously
from ER vesicles.
Cisternae move through the
stack from cis to trans and finally
break up
into transport vesicles at the
trans face.
Vesicle transport model:
Cisternae remain fixed. Both
membrane and content move
from the cis to the trans
cisternae in transport vesicles.
Unit 7
Endocytosis
- protein binds receptors
on the cell surface and
is internalized in
clathrin-coated vesicles.
- the vesicles lose their
coats and fuse with
endosomes.
- Cargo protein separates
from its receptor in
endosome.
- protein is transferred to a lysosome and degraded to
release free cholesterol
- protein receptors return to the plasma membrane via
transport vesicles
Phagocytosis
1. Recognition between plasma
membrane receptors and bacteria.
2. pseudopodia surround bacteria
3. membrane fusion and vesicle
formation
4. internalized as early endosome,
pH change, becomes late
endosome, fuses with primary
lysosome where digestive
enzymes are activated
5. becomes secondary lysosome,
6. bacteria is digested,
macromolecules diffuse across
lysosomal membrane
Pulse-Chase Autoradiography
An experimental approach to
observing protein secretion
What is Autoradiography?
• Autoradiography is the
use of radioactively
labeled molecules to
look at cell processes.
• A labeled molecule can
be located because its
radioactivity develops
the silver grains on a
photographic emulsion.
“Tailing” a labeled molecule
• Allows us to observe the
movement of molecules
through the cell over
time.
• Cellular pathways are
revealed as the progress
of molecules is
monitored.
The “Pulse” and the “Chase”
• The "Pulse" consists of radioactive material
added for a very brief period and then
washed away.
• Then the “Chase” begins- non-radioactive
molecules are added.
• This creates a group of labeled molecules,
with unlabeled molecules in front and
behind, that move through a particular
metabolic pathway.
Now for a demonstration!
• Stomach cells which secrete digestive
enzymes (zymogen) are supplied with
radioactively labeled amino acids.
• After a brief period -the “Pulse” - the excess
labeled amino acids are washed away.
• The cells are then supplied with unlabeled
amino acids -the “Chase”
And in the TEM we see…
Three minutes after pulse…
Labeled amino acids
(incorporated into
newly synthesized
proteins) are
localized around
RER.
The “squiggles” show
radioactivity.
Seven minutes after pulse…
• The majority of
the newly
synthesized
proteins have
moved to the
periphery of the
Golgi complex.
• Arrows indicate
periphery of Golgi
complex.
37 minutes after pulse…
• Labeled proteins are
concentrated over
secretory vesicles
called condensing
vacuoles (CV).
• Arrows indicate
periphery of Golgi
complexes.
• Secretory vesicles
containing zymogen
are marked Z.
117 minutes after pulse…
• Radioactivity is
mainly localized
over secretory
vesicles containing
zymogen.
• Some labeled
protein has already
been secreted into
the lumen (L) .
Where are they now?
(CV)
(Z)
• Samples are
taken after
various time
periods and
the location
of the labeled
molecules is
identified.
Got it?
Feed the cell a pulse of
radioactively labeled
molecules and see
what it does with
them.
So here are a few
questions…
1
2
3
4
1. PULSE: Cell is exposed to
radioactively-labelled
nucleotides (green) for 3
minutes. The nucleotides
are taken up by the
nucleus.
1
2
3
4
2. CHASE: The cell is
exposed to an excess of
non-radioactive
nucleotides. After five
minutes of chase, some
radioactive molecules are
found in the cytoplasm.
1
2
3
4
3. 15 MINUTES: Most of the
radioactivity has moved
from the nucleus to the
cytoplasm after 15 minutes
of the chase.
1
2
3
4
4. 30 MINUTES: After 30
minutes, all of the
radioactive label is found
outside the nucleus.
1) What
macromolecule is
being studied in this
experiment? Why?
A.
B.
C.
D.
E.
Polypeptides
Lipids
DNA
RNA
Carbohydrates
2. Where is this
macromolecule being
synthesized?
A.
B.
C.
D.
E.
Nucleus
Golgi
Rough ER
Cytosol
Smooth ER
3. After thirty
minutes, is the
macromolecule
still being
synthesized?
4) Which is the best explanation
for the observations?
A. Synthesis doesn't continue as
there is no more labeled
nucleotide.
B. Synthesis doesn't continue as the
cell is not making any more RNA.
C. You cannot 'see' that synthesis is
continuing as the nucleotides
being incorporated are not labeled
and hence can not be visualized
by autoradiography.
NEXT TUTORIAL
Structure and function of the
CYTOSKELETON
– Actin Microfilaments
– Microtubules
– Intermediate Filaments
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