ECM

plasma
membrane

endosome
secretory
vesicles

lysosome
Endomembrane
system
mitochondrion

Golgi
apparatus

chloroplast

ER

nucleus

Vesicle Budding & Trafficking

Sorting signals lead to cargo proteins being loaded
into particular vesicles - video
1) Vesicles bud via COP and clathrin proteins
2) Vesicles told to fuse to next compartment
by Rab and SNARE proteins

Vesicle Budding
Budding vesicles covered on cytosolic surface by
protein coats:
- soluble proteins that assemble on donor membrane
-often bind to receptor used for protein sorting so
they select components to be packaged into vesicle
e.g. M-6-P receptor, KDEL receptor
-function as a mechanical device to cause the
membrane to curve & form a budding vesicle
Disassemble from membrane after budding
1) COP proteins: coat proteins
2) clathrin

vesicle starting to
bud off membrane,
with the coat
protein pinching the
membrane

Fig. 8.6 video

COPII-coated vesicles - move materials forward

from ER to Golgi
COPI-coated vesicles
-transport materials from trans Golgi back
to cis Golgi- retrieval vesicles
-move materials in retrograde direction from
the Golgi back to ER

retrograde,
transportation
(going back)

COPII-coated ER
COPI-coated Golgi cisternae
Clathrin-coated: trans-Golgi network, PM
Others seen by EM

involved in proteins
going to the
endosomes and
lysosomes,
involved in
endocytosis

COPII-coated transport vesicles

Bud from ER exit sites
Dispersed throughout ER membranes
Regions without bound ribosomes
forms a sphere
ones the vesicle
buds off

Retention of ER proteins:

Cargo selection:

H3+N
KDEL
(Lys-Asp-Glu-Leu)

Figure 8.28

secreted
protein

lysosomal
enzyme
(carries
mannose 6-P)

Mannose 6-P
Receptor
video

gets solublized into

the cytoplasm

I- cell disease

secreted
protein

lysosomal
enzyme
(carries
mannose 6-P)

Mannose 6-P
receptor

-missing enzyme
responsible for mannose
phosphorylation
- lysosomal enzymes
secreted

clathrin

forms triskelion (6proteins)

assembles into basket structure
6 proteins forming
the complex

Clathrin-coated vesicles - traffic materials from

TGN to lysosomes
- also move materials from cell membrane to
cytoplasmic compartments along endocytic
pathway

moving proteins
from the golgi to
lysosomes through
endosomes

Fig. 8.20

Clathrin-coated vesicles
Adaptin Binds clathrin to membrane

- Concentrates cargo receptors

Packages cargo in clathrin-coated transport vesicles

Dynamin - Pinches off bud (transport vesicle)

goes to specific
regions

- BOTOX
video
long video

Rabs and
SNAREs are involved in
determining the
specificity of membrane
fusion events
- often occurs along
the cytoskeleton
pulls the complex
together

Fig 8.31

v- (vesicle) SNARES
bind t- (target) SNAREs

Rabs

SNARES

Copyright, , 2002, John Wiley & Sons, Inc.,

Cytoskeleton
Rabs Direct
Vesicles

SNARES mediate
Docking and
Fusion

Karp/CELL & MOLECULAR BIOLOGY 3E

Rabs

SNARES

Copyright, , 2002, John Wiley & Sons, Inc.,

Cytoskeleton
Rabs Direct
Vesicles

SNARES mediate
Docking and
Fusion

Karp/CELL & MOLECULAR BIOLOGY 3E

trans- golgi
network

NSF

Trans-SNARE
complex disassembly

Rab

Rab

v-SNARE recycled to
donor compartment
t-SNARE free to
accept new vesicle
NSF

Trans-SNARE
complex disassembly

The Rab family of small GTP-binding proteins

plays a key role in vesicle budding and fusion
reactions.
!

Rab proteins mark different organelles and

transport vesicles, so their localization on
the correct membrane is key to establishing
the specificity of vesicular transport.
!

Rab5 video

a coat protein is
attached

Need correct
combination
of Rabs and
SNAREs
Lipid group
exposed;
anchors in
membrane
Rab and
effectors
help vesicle
dock at
correct
target
membrane

active
(binds C-ter of specific Rab)

inactive

(prevents GDP
exchange until at
correct donor
membrane)

cytosol

Rab brings the vsnare and t-snares

together, by
bringing it to the
right spot

video

Vesicle fusion

From the TGN:

secretory
granule
TGN
constitutive
secretory vesicle
Plasma
membrane
lysosomes

secretory
granules

secretory
vesicles

lysosome
Q: How are proteins
sorted within the
endomembrane
system?
A: Sorting signals

Golgi
apparatus
ER
What is the default?
Secretion

Biosynthetic (secretory) pathway

- synthesis in ER or Golgi
- altered as they pass through ER & Golgi
- proteins move to PM or are secreted from
cell
Two types of secretory activity
1) Constitutive
2) Regulated

Biosynthetic (secretory) pathway

1) Constitute Secretion
- synthesis & secretion into extracellular
space occurs in continuous, unregulated
manner
- most plasma membrane and ECM components

Biosynthetic (secretory) pathway

2) Regulated Secretion
- secretory materials stored in big, densely
packed, membrane-bound secretory granules
in cell periphery
- secreted only after correct stimulus
ie. release of calcium ions from SER
eg. hormones, digestive enzymes, neurotransmitters

Figure 8.11

How do Proteins Maintain Orientation

During Secretion?

Figure 8.14

Exocytosis: The Terminal Stage of Secretion

- process of membrane fusion & content
discharge into the ECM
When cytoplasmic vesicle fuses with cell
membrane:
- luminal vesicle surface becomes part of outer
cell membrane surface
- sugars on glycoproteins face extracellularly
-soluble proteins released to
the ECM

Sorting of lysosomal enzymes:

In the cis Golgi, mannose sugars on lysosomal
enzymes get phosphorylated:
P

Mannose-6-Phosphate

Binds to M-6-P Receptor on TGN

Clathrin binds to M-6-P Receptor on cytosolic face
Vesicle pinches off containing lysosomal enzymes
Fuses with late endosomes

becomes Lysosomes

M-6-P receptors recycled

Biosynthetic/
Secretory
Pathway

bringing content
from outside the
cell inside

Endocytic
Pathway

Figure 8.2

Fig. 8.29b

Cellular Uptake of Particles and Macromolecules

materials too large to pass through membrane
uptake of transporters and signaling receptors
no longer required
uptake of extracellular materials (ligands)
uptake occurs into cytoplasmic vesicles
derived from invaginations of cell membrane
Two separate categories:
endocytosis
phagocytosis

Endocytosis
uptake of dissolved solutes, macromolecules,

signaling receptors, etc.

Receptor-mediated endocytosis (RME)
uptake of specific extracellular macromolecules

(ligands) that bind to receptors on external cell

membrane surface
take up hormones, growth factors, enzymes,
plasma proteins by binding to receptors

(b) Bulk phase endocytosis

(b) Bulk phase endocytosis

Receptormediated
endocytosis

EE

cargo brought in
and the receptors
that brought it in
will let go, when it
reaches the
destination

lysosome

H+ H+
H+

SC
=clathrin

H+
H+

H+
H+

ECV
Lysosomal
enzymes
(M6P)

LE
TGN

Endocytic Pathway (receptor-mediated)

Receptors bind ligand - undergo conformational
change, clathrin binds on cytosolic side
PM pinches in (via clathrin) to form early
endosome (EE)
Early

endosome matures into sorting

compartment (SC)
Sorting

compartment lumen acidified due to

action of membrane H+ pumps
In low pH, ligands are released from cell
membrane receptors

Endocytic Pathway (receptor-mediated)

Receptors are recycled back to cell membrane

in vesicles
Endosome

carrier vesicles (ECVs) leave SC

and fuse with late endosomes (LE)
or fuse with
lysosomes

mature into lysosomes

where contents are
digested (via acid
hydrolases)

Fig. 8. 43

Fig 8.37

Cholesterol transport

Cholesterol uptake

Endocytosis of LDL (with ApoB-100) recognized by

LDL-receptors on plasma membrane of cells

Binding triggers receptor-mediated endocytosis

44

Aggregate in coated pits

Internalized as endocytic vesicles that acidify to form

endosomes

Endosomes fuse with lysosomes

Degradative enzymes hydrolyze Cholesterol esters

Release fatty acid and free cholesterol to cytosol (for membrane

synthesis or storage)

ApoB-100 degraded; amino acids released to cytosol

LDL-receptor recycled

LDL takes
cholesterol in but
you're body makes
it anyways if you
doesn't have
cholesterol in you're
diet

Cholesterol transport

(ApoB-100)

45

Phagocytosis (cell eating)

uptake

also can be used

to kill bacteria,
any foreign
material (white
blood)

of relatively large particulate matter

(>0.5 m in diameter)
for

food, eg. unicellular heterotrophic protists

for protection, eg. macrophage, dendritic cells and

neutrophil immune cells
Receptors

on cell bind particles (bacteria, dead

cells, etc.)
Macrophage have Fc-receptor - binds to
antibodies (IgG) that coat (called opsonize)
pathogens, etc.

microphage eating
a cell thats likely to
be infected

Fig. 8.46

Phagocytosis (cell eating)

Receptors cluster into lipid rafts
Clustering signals recruitment of signaling
proteins
Src (lipoprotein)
PI3K (phosphorylates PIs)
PI3K phosphorylates PI on plasma membrane
to make PI3P

PI (Phosphatidylinositols)
PI
OH
OH
PI3P
OH
OH

O
-O
P O
O

FA

OH
P

FA

Diglyceride

PI

Inositol Ring

puts the signal on

PI3 kinase

Phagocytosis (cell eating)

PI3P recruits PH-domain containing proteins
This protein domain has a high affinity to
PI3P (ie will bind to it)
PH-proteins bind to actin proteins and cause the
underlying actin cytoskeleton to rearrange
Actin rearrangement causes large membrane
extensions which engulf particle
Particle internalized into membrane-bound
compartment called phagosome

Phagocytosis

IgG-particle
FcR

Phagocytosis

signaling protein
that
phosphorylates
other proteins

IgG-particle
FcR

allows the
attraction of the PH

PI

src

PI3K

PI3P

PH

Cytoskeletal Rearrangements

Phagocytosis and Phagolysosome Formation

bacteria engulfed

IgG-opsonized
IgG-opsonized
particle
particle

IgG-opsonized
particle
H+
lysosome
H+

H+

Phagocytosis (cell eating)

Phagosome fuses with lysosomes
phagolysosome
Acid hydrolases kill/digest bacteria
Some pathogens escape lysosomes
consequently are not killed and can proliferate in
macrophages
prevent the
invagination to
occur

eg. Mycobacterium, Salmonella

Pathogens secrete factors that interfere with Rab
proteins so lysosomes are not targetted correctly to
phagosomes

Experiment
Are phosphoinositides important for phagocytosis?
Watch a probe for these phospholipids and see
if the probe accumulates during phagocytosis in a
macrophage
What is a good probe?
PH-domains

in proteins are known to bind to

PI3P
so fuse the PH-domain to GFP
See if PH-GFP accumulates during phagocytosis
by confocal microscopy

PH domains of proteins bind PIP3

PI

PI3P
PH

PI3K

GFP

clustered at regions
where invagination
occurs

Lysosomes
- animal cell's digestive organelle
- contains ~50 different hydrolytic enzymes
made in RER
Enzymes are acid hydrolases
!

-function at acidic pH
-enzymes can hydrolyze
virtually every
macromolecule
Fig. 8.33
Range in size from 25 nm- 1 m in diameter

Lysosomes
Lysosomes are acidic- have many proton pumps
Drives protons (H+) in lumen of lysosome

H+

H+
H+
H+

Lysosomes
Lysosomes are acidic- have many proton pumps
Drives protons (H+) in lumen of lysosome
H+
H+ H+ H+
H+ H+
H+

High [H+]
= low pH
= acidic

Lysosome Functions
- digest contents brought into the cell by
endocytosis or phagocytosis = phagolysosome
- digest organelles autophagy = autophagolysosomes

Lysosome Functions
- lysosomes can fuse with plasma membrane in
a few cells to digest ECM (via exocytosis)
eg. Osteoclasts - bone cells that exocytose
lysosomes to degrade bone

Bone

Bone

Bone

H+

H+
H+

H+
H+

H+

H+

acid
hydrolase

Lysosome Functions
- lysosomes can fuse with plasma membrane in
a few cells to digest ECM (via exocytosis)
eg. Osteoclasts - bone cells that exocytose
lysosomes to degrade bone
eg. Sperm - sperm head contains specialized
lysosome called the acrosome
-during fertilization the lysosome fuses with
PM on sperm to release enzymes and digest
eggs outer covering

Lysosome Digestion
- digested contents become small by-products
- beneficial molecules transported into cytosol
- undigested and unwanted products become
residual body which either remains in cytoplasm
or is exported by exocytosis

Mannose 6 phosphate on lysosomal proteins

https://www.youtube.com/watch?v=u38LjCOvDZU

!
Secretion https://www.youtube.com/watch?v=MrHULUxAsGg
https://www.youtube.com/watch?v=V2FrQB6rX34
http://www.garlandscience.com/garlandscience_resources/
resource_detail.jsf?
landing=student&resource_id=9780815344322_CH13_QTM02
Botox: https://www.youtube.com/watch?v=dkpohXE06pg
https://www.youtube.com/watch?v=NZZ9fI3U4k0
Endocytosis: http://www.garlandscience.com/garlandscience_resources/
resource_detail.jsf?
landing=student&resource_id=9780815344322_CH13_QTM01
http://www.garlandscience.com/garlandscience_resources/
resource_detail.jsf?
landing=student&resource_id=9780815344322_CH13_QTM03
http://www.garlandscience.com/garlandscience_resources/
resource_detail.jsf?
landing=student&resource_id=9780815344322_CH13_QTM04

Copyright, , 2002, John Wiley & Sons, Inc.,

Karp/CELL & MOLECULAR BIOLOGY 3E