Download Lecture 3: Protein trafficking between cell compartments The cytosol

17/01/2017
Cell biology 2017
Protein trafficking between cell compartments
version 13/1 2017 Note – endosome vs lysosome handout
Lecture 3:
Free cytosolic
Ribosome
populations
Nucleus
ER
Golgi
Text book Alberts et al.: Chapter 12 - 14
(Topics covered by the lecture)
Protein
N
Ribosomes
Cytosol
Attached to the
endoplasmic
reticulum
C
Lysosome
A lot of reading! Focus on principles
Mitochondrion
>90 % of all membranes are part
of organelles within the cytoplasm
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Cell Biology interactive  media  ”video” or ”interactive”
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The cytosol
Viscous solution  high concentration of proteins (~400 mg/ml)
The mitochondrion - the power plant of the cell
• From Greek, mitos, thread, + chondros, granule
1 m
ATP (2)
Nucleus
2
• The mitochondrion is a double membrane-enclosed organelle
that specialize in ATP regeneration (>100 per cell)
Glucose
2.
1.
Various address tags
(without a tag cytosol)
NADH (2)
0.5 m
Pyruvat
The invaginations are
denoted cristae
 increased surface area
3.
Key processes/
components of
the cytosol
1. Translation
2. Glycolysis
+
3. Signal transduction
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Metabolic pathways of the mitochondrion
1. Intermediary
metabolism
• Reproduce by dividing
in two (binary fission)
Pyruvate
Fatty acid
Acetyl CoA
Acetyl CoA
Anim. 02.5-citric_acid_cycle.mov (1.5 min)
Krebs
cycle
NADH
2. Respiration (electron transport chain) and ATP synthesis
The origin of the mitochondrion and its genome
+
Aerobic
bacteria ”Founding”
eukaryote
• Mitochondria have circular DNA and bacteria-like ribosomes
22 tRNA genes
1.
37
genes
2.
2 rRNA genes
13 mRNA encoding genes
H+ gradient and utilization of its energy
for ATP production.
Anim. 14.3-electron_transport & 14.4-ATP_synthase
• 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
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Endoplasmic reticulum – ”network within cytoplasm”
Targeting proteins to the mitochondrion
1. Protein sorting and modification (Rough ER
)
(starting point of the “secretory pathway” of protein synthesis)
2. Lipid synthesis (Smooth ER)
Protein translocation across the
mitochondrial membranes is
mediated by proteins that form a
channel spanning both membranes
3. Detoxifications (Smooth ER, eg. P450)
4. Ca2+ storage (Smooth ER)
Translocation of mitochondrial proteins through this channel
requires proteins to be kept unfolded
Folded protein
1.
Unfolded protein
N
Signal sequence
No passage
Successful passage
Anim. 12.3-protein_import
Chaperone, keeping
the protein unfolded
in cytosol
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Protein targeting to the endoplasmic reticulum
Anim. 06.6-translation-I
3.
2.
L+i+p+i+d = Lipid
4.
8
Co-translational protein translocation
ER associated ribosome
SRP receptor
Cytosolic ribosome
Signal-recognition
particle (SRP)
Ribosome
mRNA
tRNA
ER lumen
ER signal sequence,
stretch of hydrophobic a.a.
Protein is translocated
into the lumen of the
ER co-translationally
Protein translocator
Signal sequence is cleaved by
a peptidase after completion
of translation/ translocation
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Pause in translation during localization step
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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
Note the opening of the
protein translocater,
which allows lateral
diffusion within the ERmembrane of both the
ER-signal sequence
and trans-membrane
domain
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Synthesis of multi-pass transmembrane proteins
Re-start-transfer sequence
ER entry: the first step to various destinations
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ER signal sequence (N-terminus)=
the initial ”start transfer
signal” followed by a
signal peptidase
recognition site
CStop-transfer sequence
Post office
Plasma membrane
ER
ER
SRP
Out of the cell (secretion)
Lysosome
Golgi
Secretory pathway
January 2017
Translocation stop and re-start several times,
which results in a multi-pass transmembrane protein
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Anim. 12.6-protein_translocation.mov
Proteins are glycosylated during passage
of the secretory pathway
Vesicular trafficking post ER
“Glycocalyx – a carbohydrate
zone on the cell surface”
Extracellular
Post-translational modification
by attachment of oligo-saccharides
O-linked oligo-saccharides are attached to hydroxyl
group of serine or threonine in Golgi
O
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)
Video 13.2-biosy_secret_path
Principle of vesicular transport
Nucleus
Donor compartment
1. Budding of vesicle from
donor compartment
3-10 Golgi cisterna
(containing different
sets of processing
enzymes)
Downstream
target
compartments
Golgi
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The architecture of the Golgi apparatus
Trans-face
Out of the cell
Plasma membrane
ER
Cytosol
N-linked oligo-saccharides are attached via the amide group
of asparagine in ER
H
N
ER
Proteins that keep the
Golgi cisterna together
Post office
The cytoskeleton is
used often used as
railway tracks
2. Vesicle transport
Cis-face
3. Docking and fusion of
a vesicle with its
target compartment
ER
Transport vesicles
Target compartment
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Vesicle formation in donor compartment
Different coating proteins in vesicular trafficking
3. Vesicle pinching off
Endocytosis
at the plasma
membrane
Coat:
2. Vesicle formation
Clathrin
COPI
Coat 1. Bud formation
protein
Sorting receptor
Cargo
(i.e., the protein
to be transported)
COPII
Cargo
Constricting protein complex
Lysosome
Adaptin
Sorting
receptor
Golgi
Lumen of donor compartment
ER
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Vesicle docking and fusion with target compartment
Tethering of vesicles to the correct target compartment
1. Uncoating of vesicle subsequent to ”pinching off”
Rab protein on vesicle
docks with Rab effector
on target compartment Rab protein
2. Vesicle tethering with target compartment (specificity Rab’s)
3. Vesicle docking and fusion with target compartment (SNAREs)
Rab effector
(tethering protein)
Different Rab proteins – different target compartments
1.
Compartment X
2.
3.
Compartment Y
Lumen of target compartment
Fusion of a vesicle with its target compartment
Protein trafficking in the vesicular pathway
v-SNARE
1.
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Plasma
membrane
2.
Lysosome
3.
Clathrin
Golgi
t-SNARE
1. SNARE proteins on vesicle and target
compartment interacts
COPI
Retrieval of
ER proteins
4.
(KDEL receptor)
Anterograde
transport
2. Conformational changes of SNAREs bring
the membranes closer together…..
COPII
Retrograde
transport
3. …..until they are in physical contact
4. This leads to exclusion of H2O  membrane fusion
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Endoplasmic
reticulum
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Protein trafficking: post-Golgi
Exocytosis
Regulated
Constitutive
(e.g. insulin)
The lysosome – the digestive system of the cell
Lysosomal pathway
A Primary lysosome
• Vesicles (~ 300/cell) filled with ~ 40 acid hydrolases that has
capacity to degrade more or less anything
B Endosome
• The lysosome is responsible for degradation of exogenous and
endogenous macromolecules and structures
C Secondary lysosome
The term lysosome
defines a function:
lys: digest
soma: body
pH 7.2
Lysosomes develop
from endosomes
by fusion with
vesicles carrying
lysosomal enzymes
B
A
Anim. 13.1clathrin
• The inside of the lysosome is acidic
0.2-0.5 m
H+
pH 5
H+
H+
C
Video 13.4endosome_
fusion
ATP
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H+
The pH regulates the activity of hydrolytic enzymes
Lysosome contains many types of hydrolytic enzymes
These are only active in an acidic environment
+
ADP + P
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Uptake of material from the exterior
1. Phagocytosis (“cell eating”) – specific uptake of large
(0.5 – 2 m) 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
Degradation of
endocytosed
material
Hydrolases are
inactive in ER and
Golgi (pH ~7)
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1.
Hydrolases are active
in the acidic lumen of
the lysosome
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Hydrolases: proteases, nucleases, phosphatases etc etc.
video 13.5 phagocytosis .mov
Three routes to the lysosome
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%)
Phagocytosis
1.
Endocytosis
4.
2.
4.
5.
4.
ER
3.
Autophagy
1. Phagosome
2. Endosome
4. Primary lysosome
Anim. 13.3-receptor_endocytosis
3.
2.
>10-fold more internal
membranes than
plasma membrane
3. Autophagosome
5. Secondary lysosome
(Note: vesicle fusion with endosome)
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% = volume of a liver cell
Nucleus (6%)
ER (12%)
Golgi (3%)
**
Ribosomes
**
Lysosome (1%)
Mitochondrion (22%)
Endosome (1%)
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