Endocytosis - Exocytosis
László KŐHIDAI,
Med. Habil. MD, PhD., Assoc. Prof.
Department of Genetics, Cell- and
Immunobiology
Semmelweis University
2008
Endocytosis
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Phagocytosis – solid
Pinocytosis – liquid (general)
Endocytosis:
• Uptake of substances
• Transport of protein or lipid components of
compartments
• Metabolic or division signaling
• Defense to microorganisms
Phagocytosis (1)
Predominant cells:
• unicellular cells
• macrophages
• osteoslats
• throphoblasts
Functions:
• uptake of food
partickles
• immuneresponses
• elimination of aged
cells (RBC)
Phagocytosis (2)
Required:
• signal
• membrane receptor (Fc receptor for Ab)
• formation of pseudopodium
• cortical actin network
The formed vesicle: phagosome (hetero-; auto-)
Endocytosis
• Clathrin-coated vesicles
• Non-clathrin coated vesicles
• Macropinocytosis
• Potocytosis
Clathrin coated
pits/vesicles
Function of clathrin coated vesicles
Receptor mediated endocytosis
• Selective uptake of molecules
(low environmental conc.)
• Membrane receptors
• Concentration of ligand (1000x)
Components of coated vesicles
Receptor-mediated endocytosis of LDL
Sorting signals of secreted and membrane
proteins to transport vesicles
Selective incorporation
of membrane proteins
Into the coated vesicles
dER
TGN
LRO
LE
EE
L
RE
-clathrin
-AP-2
-AP180
-actin
-synaptotagmin
-PIP2
-dynamin
Endosomal-Lysosomal compartment
Structure
• tubular, vesicular
• acidic pH - vacuolar H+ ATP-ase - proton pump
• early-endosome (EE) and late-endosomes (LE)
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•
and lysosomes (L)
EE pH= 6; LE pH=5
in EE no lysosomal membrane proteins or
enzymes (in contrast LE)
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Endosomal-Lysosomal compartment
Function
sorting
transport
degradation
removal of clathrin layer
formation of EE
in the EE:
• dissociation of receptor-ligand complex - receptorrecycling (e.g. LDL, transferrin)
• receptor-ligand complex transported together - receptor
down regulation (e.g. EGF)
Pathway of LDL
-insulin or other
hormones –
in receptor
mediated
endocytosis
Fate of LDL
internalized by
receptor-mediated
endocytosis
The transferrin-cycle
Partcipating components in budding
of coated vesicles
Late endosome
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early endosomes, TGN and autophagosomes feed
late endosomes
lysosomal enzymes M-6-P signal is changed, the
phosphate group is cleaved - receptors can not
bind enzymes
the enzyme content of vacuoles is in the lumen
lysosomes
Dissociation of receptor-ligand complex in
late endosomes
De Duve, Ch.
Nobel-prize - 1974
Lysosomes (TEM)
Lysosomes
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enzymes - acidic hydrolases
e.g. protease, nuclease, glycosidase, phosphatese
more than 40 types of enzymes
membrane proteins - highly glycosilated protects
from the enzymes
transport molecules of the membranes transports the products of proteolytic cleavage into
the cytoplasm
the waste products are released or stored in the
cytoplasm (inclusion - residual body)
LAMP = lysosome associated membrane proteins
• integrant membrane proteins of
the lysosome
• LAMP-2 – tarnsport of cholestero
• LAMP-2 defficiency- autophagy
www.helsinki.fi/bioscience/biochemistry/eskelinen
Autophagy - Autophagosome
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intake of own components
regulates the number of organells
toxic effects can also induce it
Formation of autophagosome
www.helsinki.fi/bioscience/biochemistry/eskelinen
E
Non-clathrin coated vesicles
• There is no receptor or clathrin in the membrane
• The uptake of substances is less selective
• Primairly liquide-phase endocytosis
Macropinocytosis
• Ruffling of the surface membrane forms inclusions
• These „vacuoles” have no membrane
• Size 0.2-5 mm - the mass/surface ratio is very good
Significance:
• Liquide-phase pinocytosis
• Taking probes from the
environment
– antigene recognition
in macrophages
Film produced by F. Vilhardt and M. Grandahl.
Caveolae
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50-80 nm, bottle-like infoldings of the surface
membrane
endothels, adipocytes
caveolin
potocytosis - caveolae close but not internalized,
the materials enter the cytoplasm by
a special carrier molecule
e.g. vitamine B4
some other caveolae enter the cell !!!
Caveolae
Caveolin oligomers and caveolae
assembly
33 AA
44 AA
C
101 AA
N
Dynamincs of caveolae-formation
Functions of dynamin
Clathrin-mediated endocytosis
Membrane
retrieval
Endosometo-Golgi
transport
Secretory
vesicle
formation
in TGF
Caveolae
Fluid phase
endocytosis
Dynamin in the cell
Structure of dynamin
Interaction with
membranes
Activation of
GTP-ase domain
Interaction with
cytosceleton
Dynamin requires GTP hydrolysis to pinching off
coated vesicles
• The not-hydrolysable GTP-gS is added
• Dots represent binding of anti-dynamin antibodies
• The long neck shows that however the coated pit was formed,
in the absence of GTP hydrolysis its pinching off is absence
Carrier mediated proteolysis
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some molecules can enter lysosome directly from
the cytoplasm
the signal of entry: KFERQ (Lys-Phe-Glu-ArgGln)
Proteasome
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non-lysosomal cleavage of proteins
cylindric, multienzyme complex
parts: ATP binding-, substrate binding-, regulator-domain
location: close to the external part of ER-translocon
ubiquitin - degradation-signal - is required
the non-properly folded or damaged proteins
regulator - eliminator - role e.g. cyclins
cystic fibrosis - Cl- fac. transp. is affected as the responsible
membrane protein is broken down in proteosome
Proteasomes
Ubiquitation - proteasome
„Exocytotic” processes
The mannose-6-P pathway and lysosomal enzymes
Exocytosis in TEM
Apical and basolateral targeting in epithelial cell
Transcytosis
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the ligands walk around the endosomal
compartment
ligands transported from one surface to the other
e.g. immunoglobulins of the colostrum cross the
intestinal epithelium by transcytosis
Release of neurotransmitters