Secretory pathway

Report
Cell biology 2014 (Revised 23/1 -14)
Note handout concerning endosomes/lysosomes
Lecture 3:
Alberts et al
5th edition
Chapter 12
Chapter 13
699-702
713-714
723-739
743-745
749-751
754-800
809
Chapter 14
A lot of reading!
Focus on general
principles and topics
highlighted in the lecture
synopsis
813-819
1
Cell Biology interactive  media  ”video” or ”interactive”
Protein trafficking between cell compartments
Ribosome
populations
Nucleus
ER
Golgi
Cytosol
Ribosomes
Free cytosolic
Attached to the
endoplasmic
reticulum
Protein
N
C
Lysosome
Mitochondrion
>90 % of all membranes are part
of organelles within the cytoplasm
Various address tags
(without a tag cytosol)
2
3
The cytosol
Viscous solution  high concentration of proteins (~400 mg/ml)
Glucose
2.
1.
ATP
Nucleus
NADH
Pyruvat
3.
Key processes/
components of
the cytosol
1. Translation
3. Signal transduction
2. Glycolysis
The mitochondrion - the power plant of the cell
• From Greek, mitos, thread, + chondros, granule
• The mitochondrion is a double membrane-enclosed organelle
that specialize in ATP regeneration (>100 per cell)
1 mm
0.5 mm
• Reproduce by dividing
in two (binary fission)
The invaginations are
denoted cristae
 increased surface area
+
4
Metabolic pathways of the mitochondrion
Pyruvate
1. Intermediary
metabolism
Acetyl CoA
Fatty acid
Acetyl CoA
Krebs
cycle
Anim. 02.5-citric_acid_cycle.mov (1.5 min)
NADH
2. Respiration (electron transport chain) and ATP synthesis
2.
1.
Generation of a H+ gradient
and utilization of its energy
for ATP production
Anim. 14.3-electron_transport & 14.4-ATP_synthase
(Alberts et al Fig. 14-10)
The origin of the mitochondrion and its genome
+
Aerobic
bacteria ”Founding”
eukaryote
• Mitochondria have circular DNA and bacteria-like ribosomes
22 tRNA genes
37
genes
2 rRNA genes
13 mRNA encoding genes
• Mitochondria are only inherited from the mother
• Most of the mitochondrial proteins are encoded in the
nucleus and have to be imported from the cytosol
6
Targeting proteins to the mitochondrion
Protein translocation across the
mitochondrial membranes is
mediated by proteins that form a
channel spanning both membranes
Translocation of mitochondrial proteins through this channel
requires proteins to be kept unfolded
Folded protein
Unfolded protein
N
Signal sequence
No passage
Anim. 12.3-protein_import
Successful passage
Chaperone, keeping
the protein unfolded
in cytosol
7
Endoplasmic reticulum – ”network within the cell”
1. Protein sorting and modification (Rough ER
)
(starting point of the “secretory pathway” of protein synthesis)
2. Lipid synthesis (Smooth ER)
3. Detoxifications (Smooth ER, eg. P450)
4. Ca2+ storage (Smooth ER)
1.
3.
2.
L+i+p+i+d = Lipid
4.
8
Protein targeting to the endoplasmic reticulum
Anim. 06.6-translation-I
Cytosolic ribosome
ER associated ribosome
SRP receptor
Signal-recognition
particle (SRP)
Ribosome
mRNA
tRNA
ER lumen
ER signal sequence,
stretch of hydrophobic a.a.
Protein translocator
Pause in translation during localization step
9
Co-translational protein translocation
Protein is translocated
into the lumen of the
ER co-translationally
Signal sequence is cleaved by
a peptidase after completion
of translation/ translocation
10
Integration of a transmembrane protein into ER
C
N
Translocation is
initiated but stops at a
hydrophobic ~15 aa
sequence termed stoptransfer signal
However,
translation
continues
Translation complete
the stop-transfer
signal sequence
integrates into
the ER membrane
11
Note the opening of the
protein translocater,
which allows lateral
diffusion within the ERmembrane of both the
ER-signal sequence
and trans-membrane
domains
12
Synthesis of multi-pass transmembrane proteins
Re-start-transfer sequence
ER signal sequence (N-terminus)=
the initial ”start transfer
signal” followed by a
signal peptidase
recognition site
CStop-transfer sequence
SRP
Translocation stop and re-start several times,
which results in a multi-pass transmembrane protein
Anim. 12.6-protein_translocation.mov
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Entry into ER is in most cases only
the first step to a final destination
Post office
ER
ER
Out of the cell (secretion)
Plasma membrane
Lysosome
Golgi
Secretory pathway
14
Proteins are glycosylated during passage
of the secretory pathway
“Glycocalyx – a carbohydrate
zone on the cell surface”
Post-translational modification
by attachment of oligo-saccharides
Extracellular
Cytosol
N-linked oligo-saccharides are attached via the amide group
of asparagine in ER
H
N
ER
O-linked oligo-saccharides are attached to hydroxyl
group of serine or threonine in Golgi
O
Golgi
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Vesicular trafficking post ER
Post office
Out of the cell
Plasma membrane
ER
Golgi
Lysosome
Secretory pathway
Transport from ER to Golgi, within Golgi, and from Golgi to
either lysosomes or cell surface is carried out by transport
vesicles (liposomes made of phospholipids)
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Video 13.2-biosy_secret_path
The architecture of the Golgi apparatus
Proteins that keep the
Golgi cisterna together
Nucleus
3-10 Golgi cisterna
(containing different
sets of processing
enzymes)
Trans-face
Downstream
target
compartments
Cis-face
ER
Transport vesicles
17
Principle of vesicular transport
Donor compartment
1. Budding of vesicle from
donor compartment
The cytoskeleton is
used often used as
railway tracks
2. Vesicle transport
3. Docking and fusion of
a vesicle with its
target compartment
Target compartment
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Vesicle formation in donor compartment
3. Vesicle pinching off
2. Vesicle formation
Coat 1. Bud formation
protein
Sorting receptor
Cargo
(i.e., the protein
to be transported)
Constricting protein complex
Lumen of donor compartment
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Different coating proteins in vesicular trafficking
Endocytosis
at the plasma
membrane
Coat:
Clathrin
COPI
COPII
Lysosome
Adaptin
Cargo
Sorting
receptor
Golgi
ER
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Vesicle docking and fusion with target compartment
1. Uncoating of vesicle subsequent to ”pinching off”
2. Vesicle tethering with target compartment (specificity Rab’s)
3. Vesicle docking and fusion with target compartment (SNAREs)
1.
2.
3.
Lumen of target compartment
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Tethering of vesicles to the correct target compartment
Rab protein on vesicle
docks with Rab effector
on target compartment Rab protein
Rab effector
(tethering protein)
Different Rab proteins – different target compartments
Compartment X
Compartment Y
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Fusion of a vesicle with its target compartment
v-SNARE
1.
2.
t-SNARE
1. SNARE proteins on vesicle and target
compartment interacts
3.
4.
2. Conformational changes of SNAREs bring
the membranes closer together…..
3. …..until they are in physical contact
4. This leads to exclusion of H2O  membrane fusion
23
Protein trafficking in the vesicular pathway
Plasma
membrane
Lysosome
Clathrin
Golgi
COPI
COPII
Retrieval of
ER proteins
(KDEL receptor)
Retrograde
transport
Anterograde
transport
Endoplasmic
reticulum
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Protein trafficking: post-Golgi
Exocytosis
Constitutive
Lysosomal pathway
A Primary lysosome
Regulated
B Endosome
(e.g. insulin)
C Secondary lysosome
The term lysosome
defines a function:
lys: digest
some: body
Lysosomes develop
from endosomes
by fusion with
vesicles carrying
lysosomal enzymes
B
A
C
Anim. 13.1clathrin
25
The lysosome – the digestive system of the cell
• Vesicles (~ 300/cell) filled with ~ 40 acid hydrolases that has
capacity to degrade more or less anything
• The lysosome is responsible for degradation of exogenous and
endogenous macromolecules and structures
• The inside of the lysosome is acidic
pH 7.2
H+
0.2-0.5 mm
H+
pH 5
H+
ADP + P
ATP
H+
26
The pH regulates the activity of hydrolytic enzymes
Lysosome contains many types of hydrolytic enzymes
These are only active in an acidic environment
+
+
Degradation of
endocytosed
material
Hydrolases are
inactive in ER and
Golgi (pH ~7)
Hydrolases are active
in the acidic lumen of
the lysosome
Hydrolases: proteases, nucleases, phosphatases etc etc.
27
Uptake of material from the exterior
1. Phagocytosis (“cell eating”) – specific uptake of large
(0.5 – 2 mm) particles, primary by immune cells
2. Receptor-mediated endocytosis - specific uptake of molecules
3. Non -specific endocytosis, pinocytosis (“cell drinking”) - anything
small in the extracellular fluid is taken up indiscriminately
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1.
2.
3.
Three routes to the lysosome
x
Phagocytosis
1.
Endocytosis
4.
2.
4.
5.
4.
ER
3.
Autophagy
1. Phagosome
2. Endosome
4. Primary lysosome
Anim. 13.3-receptor_endocytosis
3. Autophagosome
5. Secondary lysosome
(Note: vesicle fusion with endosome)
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Summary: cellular organelles and trafficking
3 types of protein transport
A. Gated (nuclear pores)
B. Across membranes**
(translocation channels)
C. Vesicle
(budding and fusion)
Cytosol
(54%)
>10-fold more internal
membranes than
plasma membrane
% = volume of a liver cell
Nucleus (6%)
ER (12%)
Golgi (3%)
**
Ribosomes
**
Lysosome (1%)
Mitochondrion (22%)
Endosome (1%)
30

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