The Endomembrane System

morphine
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(Taxol)
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rotundone
indigotin
salvinorin A
nicotine
The Endomembrane System
Eukaryotic cells are
compartmentalized.
Materials are synthesized and
delivered in a controlled
manner.
Endomembrane:
Endoplasmic reticulum (ER)
Golgi apparatus
Vacuoles
Secretory vesicles: endosomes,
lysosomes, granules, plasma
membrane.
Membrane-bound compartments
of the cytoplasm
Transport vesicles
Transport vesicles
Exocyctic movement
(biosynthetic/secretory):
Materials move outwards
starting from the nucleus
and progressively moving
to the plasma membrane
The processes involve
secretory vesicles/granules.
Movement direction # 1: Exocytic
Movement direction # 1: Exocytic
Secretion of biosynthetically produced compounds can
occur in two ways:

1. Constitutive secretion
Material is synthesized, transported, and discharged
from the cell in a continual manner.
Examples: extracellular matrix components

2. Regulated secretion
Material is stored in membrane-bound
compartments and discharged only in response to a
signal or stimulus.
Examples: hormone release in exocrine and/ or
endocrine cells.
Exocyctic movement
(biosynthetic/secretory):
Materials move from the
plasma membrane to
compartments.
The processes involve
endosomes and/orly
Sorting signals dictate
where material is
transported.

Movement direction # 2: Endocytic
ENDOPLASMIC RETICULUM
Endoplasmic reticulum (ER)
Smooth ER
Rough ER
Ribosomes
Nuclear
envelope
Smooth endoplasmic reticulum (SER) are extensively developed in a
number of cell types.
Functions include:
Synthesis of steroids and hormones (estrogen, testosterone, etc.)
Detoxification of environmental compounds
Oxygenases in liver
Ca
2+
storage
Ca
2+
-ATPase pumps Ca
2+
into the lumen
Sarcoplasmic reticulum in skeletal muscles releases Ca
2+
during muscle
contraction

Smooth endoplasmic reticulum (SER)
Synthesis of membrane lipids
Phosphoglyceride
Glycolipids and sphingosines (finished in the Golgi apparatus)
Synthesis of secreted extracellular proteins
Soluble antibodies, neutrotransmitters, serum albumin proteins
Synthesis of integral membrane proteins
Protein folding/insertion
Chaperones
Quality control of proteins
Elimination of misfolded proteins.
Post-translational modification of proteins (eukaryotes)
Early stage of protein glycosylation
Synthesis of GPI-anchored protein
Proteolytic cleavage of proteins.

Rough endoplasmic reticulum (RER)
The RER (ER) consists of a
single membrane.
RER is the starting point for
excreted protein synthesis:
All protein synthesized in the
RER is shuttled to its final
destination trafficked to RER
lumen
Protein is targeted to the
secretory pathway by an amino
acid sequence found in the
nascent (new) protein
RER: Protein synthesis
Importance of the signal
sequence was shown
experimentally by:

1. Removal of the signal
sequence from proteins that
are excreted caused the
protein to stay inside the cell.

2. Addition of the signal
sequence to non-secreted
proteins caused them to be
discharged from cell.
mRNA
2
1
RER
RER
Plasma membrane
cytoplasm
ribosome
Signal sequence: amino acid sequence that specifies
the target of the protein signal is generally located at
the amino- (-NH
2
) terminus.

N- terminus signal sequences are generally
proteolytically cleaved off the final protein after arriving at
their designated location.
Not all signal sequences are encoded at the N- terminus
internal sequences are also recognized that are not removed.

Amino acid signal sequences are recognized by signal
recognition particles (SRP).
SRP binds to the a.a. signal sequence as it comes off of the
ribosome
SRP is a ribonucleoprotein complex composed of:
1 RNA molecule (~30 nucleotides)
6 proteins

Initiation of protein synthesis for all proteins
starts in the cytoplasm on free ribosomes.
When the signal peptide sequence is
exposed off the ribosome it is bound by
the SRP translation stops.
The ribosome will then bind translocons
on the cytoplasmic surface of the RER
membrane (Targeting assisted by SRP
cytosol receptors).
ER lumen
ER membrane
Signal peptide on
nascent protein
mRNA
1
translocon
Synthesis of secreted proteins:
Step 1
Binding of the complex to the RER
membrane involves SRP receptor (binds
SRP) and translocon (binds ribosome):
GTPase activity of the SRP and SRP
receptor hydrolyze GTP GDP + P
i
= G
proteins
This energy is used to bind the ribosome
to the translocon and release SRP.

BiP
3
2
GDP
+ P
i
GTP

BiP or other
chaperone
Synthesis of secreted proteins:
Steps 2 + 3
The release of SRP from the ribosome-
nascent chain permits translation of the
protein to continue:
Signal sequence inserts into the
translocon channel and binds the interior.
Interaction between the signal sequence
and interior of the channel causing the
plug to move channel opens.
A signal peptidase within lumen of the
RER cleaves the signal from the peptide
chain.
BiP
4
BiP
3
Synthesis of secreted proteins:
Steps 3 + 4
Net amino acid charges (+ or -) at either end of each
transmembrane (TM) segment bias the direction of insertion =
positive-inside rule
Based on the charge of the exposed regions of TM segment
can adopt a different topology.
The hydrophobicity of the TM segment helps retain it in the
bilayer.
Insertion of integral membrane proteins
Glycolytic processes occur on both sides of the ER membrane:
Core sugar moieties are built primarily on cytoplasmic side.
Protein glycosylation occurs inside the ER lumen.
Glycosylation in the ER
The first stage of glycosylation for most proteins generally
involves N-acetylglucosamine (GlcNAc) and mannose
(Man) sugars.
Sugars are built onto a lipid carrier, dolichol-phosphate (DP),
one at a time on the cytoplasmic side of the RER membrane.
Sugars are transferred from UDP and GDP carriers to DP-
sugar chain by membrane bound enzymes called
glycosyltransferases recognize and transfer a single
particular type of sugar



ER lumen
ER
membrane
PP P
Dolichol-Phosphate
PP PP PP
Flip to ER
lumen
2UDP
UMP
+
UDP
CDP CMP
GDP GDP
4GDP
GDP
glycosyltransferases
Glycosylation in the ER: Core glycosylation
Transfers a core GlcNAc-Man sugar complex synthesized
in the ER to Asn amino acids on nascent polypeptide
chains.
Targets the sequence Asn-X-Ser/Thr or [NX(S/T)]
ER lumen
ER membrane
PP
Asn
PP
N
PP
N-linked
glycosyltransferase
Glycosylation in the ER: N-linked glycosylation
Examples of
mitochondrion-
targeted proteins

(Lodish, Berk, Zipursky. Molecular
Cell Biology. 2000)
Misfolded proteins need to be targeted for destruction
otherwise they can cause serious problems for the cell
chaperones.
All misfolded proteins are destroyed in the cytoplasm
by proteasomes need to have mechanisms to
identify and remove these proteins from the ER lumen
Calnexin: chaperone that recognizes misfolded
glycoproteins uses glucose as a signal
Binding immunoglobulin protein (BiP) +
membrane sensor proteins: chaperones that
recognize misfolded proteins
Quality control in the ER
Destruction of misfolded
proteins:
Accumulation of misfolded
proteins triggers the
unfolded protein
response (UPR).
Sensors in the ER are
kept inactive by the
chaperone BiP but if
misfolded proteins are
accumulated, BiP
molecules are incapable
of inhibiting the sensors.
Activated sensors send
signals to trigger proteins
involved in destruction of
misfolded proteins.
PERK
ATF
6
-chaperones
-transporters
-degradation
A model of the
mammalian
unfolded protein
response (UPR).
Misfolded proteins could be dangerous!!!
GOLGI APPARATUS
Golgi apparatus: supply chain management
Products travel in transport
vesicles from the ER to the
Golgi apparatus.
One side of the Golgi apparatus
functions as a receiving dock
for the product and the other as
a shipping dock.
Products are modified as they
go from one side of the Golgi
apparatus to the other and
travel in vesicles to other sites.
Camillo Golgi
(18431926)
Golgi apparatus
Golgi
apparatus
Transport
vesicle from
the Golgi
Shipping
side of Golgi
apparatus
Transport
vesicle
from ER
Receiving side
of Golgi
apparatus
1
2
3
4
4
cis
trans
Golgi complex is made up of:
cis- Golgi network (CGN)
cis- Golgi
medial- Golgi
trans- Golgi
trans- Golgi network (TGN)


Vesicles fuse with the endoplasmic reticulum
Golgi intermediate compartment (ERGIC) and
release contents in the lumen
ERGIC network is a network of large vesicles and
interconnected tubules that span the region between
the ER and the cis-Golgi.

Proteins move from RER to Golgi apparatus
through the:
RER ERGIC cis medial trans TGN

Sorting of proteins occurs in the TGN for delivery
to the plasma membrane or lysosome.

The movement of proteins from RER to cis-Golgi
is accomplished by vesicular transport.

ER sites devoid of ribosomes are the
sites where initial transport vesicles
form through budding.

These vesicular- tubular carriers
(VTC) carry encapsulated materials
away from the ER towards the ERGIC
and cis-Golgi
Movement of VTC occurs along
microtubule tracks.

RER lumenal proteins have a C-
terminal KDEL retention sequence.
KDEL: Lys-Asp-Glu-Leu
Higher affinity for KDEL signals in cis-
Golgi network than in RER.
KDEL receptors concentrate proteins
into vesicles returning to RER.

E
R
G
I
C

Golgis major functions
1. Further glycosylation and processing of glycoproteins:
- O-linked glycosylation.
- Single sugars added to serine, threonine and
hydroxylproline also to core oligosaccharides.
- Sugars added by glycosyltranferases are specific to for
every compartment of the Golgi apparatus.
- Trimming/ removal of sugars by glycosidases.

2. Modification of mannose to mannose-6-phosphate by
lysosomal enzymes (in the cis-cisternae).

3. Sorting of proteins to plasma mebrane or lysosome (in
the trans-Golgi network).

medial Golgi
medial
Golgi
All glycosylation occurs in Golgi cisterna
cis Golgi cis Golgi-
medial Golgi
1
mannose
N- acetylglucosamine
galactose
2 3 4
medial Golgi-
trans Golgi
trans Golgi
sialic acid
fucose
trans Golgi
5 6 7
8
medial Golgi
Asn Asn Asn Asn
Asn Asn
Asn Asn
Model 1: Vesicular
transport:
Vesicles move from one
cisterna to the next.
Cisternae remain fixed and
cargo moves in vesicles
Models of protein
movement in the Golgi
Nucleus
RER
Golgi
ERGIC
Plasma
membran
e
Vesicular transport model
Model 2: Cisternal maturation
Composition of cisternae
changes as cargo moves
through Golgi.
Cargo is carried in cisternae,
while resident enzymes cycle
back to a previous cisterna
by vesicle transport carriers
(VTC).
Models of protein
movement in the Golgi
Nucleus
RER
Golgi
ERGIC
Plasma
membrane
Cisternal maturation model
Evidence for cisternal maturation:

Blocking VTC movement from ER and ERGIC
using drugs or temperature sensitive mutants
show that the Golgi complex disappears over time.

Materials produced in the ER and travel through
the Golgi have been shown to remain in the Golgi
cisternae and never within Golgi- associated
transport vesicles.

Vesicles can move in both forward (anterograde)
and reverse (retrograde) directions.

Golgi cisternal composition changes over time.
Vesicle transport is important for:
1) Anterograde movement from RER to cis-Golgi
network
2) Retrograde movement from cis-Golgi network
to RER No anterograde!
3) Retrograde movement from trans-and medial-
Golgi No anterograde!
4) Anterograde movement to trans-Golgi network
to lysosome and to plasma membrane
G- proteins such as Sar1 are recruited to the vesicle forming
ER membrane powered by GTP hydrolysis.
Regulatory role includes bending the ER membrane
(curvature) and recruiting other Sec proteins to assist in
vesicle formation.
Sar1 Sar1
Sar1
2GTP
2GDP
GDP
GDP
GTP
Guanine exchange
factor (GEF)
Sar1 a-helix inserts
into cytosolic leaflet
of ER membrane
ER lumen
GEF protein
accumulation
starts vesicle
budding
protein
recruitment
Once Sar1
inserts into
the leaflet it
begins
recruiting
other COPII
proteins
Low membrane
curvature
cytoplasm
Vesicle formation step # 1:
G-proteins initiate vesicle coat formation
As coat is assembled, the membrane is already shaped into
a sphere.
These proteins can identify a particular membrane due to
their affinity for cytosolic portions of integral membrane
proteins that reside in the recipient membrane.

COPII proteins assemble sequentially stimulated to attach
Sar1 as multimers dimers (protein pairs)
Sec23
Sec24
Sec13
Sec31
Cargo receptor
Cargo protein
COPII proteins
COPII proteins
Vesicle formation step # 2:
Coat proteins bind to membranes
lumen
SNARE = SNAP receptor
SNAP = soluble NSF attachment protein
V-snare on vesicles and t-snare on acceptor membranes interact.
Interaction distorts lipid bilayers for membrane fusion.
Rab proteins (lipid anchored G-proteins) assists in target membrane
recognition and trafficking.

4 stranded
a- helix
bundle
forms
SNAP-25
Transport
vesicle
target membrane
Docking
Tethering
v-snare
t-snares
lumen
Transport
vesicle
target membrane
Rab + GTP
Tethering
proteins
Vesicle formation step # 2:
Interaction with SNAREs
Types of coat proteins
1. COPII: anterograde transport from
RER to Golgi

2. COPI: retrograde transport
through cisternae of the Golgi
Transport of KDEL receptors back to
ER KKXX signal

3. Clathrin: transports vesicles
between TGN to lysosome


TGN sorts lysosomal proteins from secreted/plasma
membrane proteins.
It is essential to remove hydrolytic lysosomal enzymes away
from secretory proteins.
Mannose-6-phosphate (M6P) acts as a signal for targeting
lumenal proteins to lysosome.
Trans-Golgi network (TGN)
Final destination:
lysosome vs. plasma
membrane

1. Early or late become
lysosomes
Clathrin dependent
secretion

2. Final secretory vesicle
for delivery to the
plasma membrane
Vesicles lack clathrin
coating
Rab dependence
LYSOSOME
Functions of lysosomes
1
2
3
3. Controlled
Uptake of
nutrients
1. Digestive
2. Autophagic
1. Digestive
Optimal pH for function is low (pH 4.6 - 5.0)
H
+
- ATPase activity (100-1000 times cytoplasm acidity)
Glycosylated interior (inner leaflet) protects
compartment from pH damage
Enriched with ~40 types of hydrolytic (degradative)
enzymes

2. Controlled Uptake Regulator
Endocytic particles (or bacteria) form endosomes which
are routed to the lysosome for degradation
Some bacteria target and happily live in endosomes eg. Coxiella
burnetti (Q fever)

3. Autophagic (Micro/ Macro types)
Organelle (macro) and ribosome (micro) turnover is
essential to remove damaged or malfunctioning cell
components (eg. mitochondria or chloroplasts)
Digestive
enzymes
Lysosome
Food vacuole
Plasma membrane
Digestion
Lysosome
Vesicle containing
damaged mitochondrion
Digestion
Mannose-6-phosphate (M6P) is added onto lysosomal
proteins in the cis-Golgi (two step reaction) permits their
identification later.

M6P is recognized by the M6P receptor (MPR) in the
TGN which sorts these proteins away from secreted
protein
Patients with I- cell disease are deficient in the enzymes that
convert mannose to M6P, or lack proper M6P receptors results
in lysosomes filled with undegraded cell structures/molecules

At TGN, lysosomal proteins are packaged into clathrin-
coated vesicles for transport to the lysosome
Mechanism for sorting lysosomal proteins
Lysosomal sorting using
clathrin coated vesicles
(CCV)
1
2
3 4
Cyto
TGN
Endocytosis involves the uptake of proteins and
other macromolecules at the plasma membrane.

Bulk materials are taken up by the cell in two
ways:
Within the membrane Proteins are concentrated
during uptake (receptor mediated endocytosis).
Within the fluid phase No increase in the
concentration of the molecules
Pinocytosis (cell drinking)
Phagocytosis (cell eating)
Lysosomes in endocytosis
1) Internalize nutrients:
- Low density lipoprotein (LDL) (cholesterol)
- Fe
3+
TRANSFERRIN

2) Internalize molecules for storage:
- Vitellogenin synthesis in liver is transported via
blood and taken up by oocytes that have vitellogenin
receptors

3) Removal of surface receptors:
"down-regulation" of receptors after stimulus
Lysosomes in endocytosis:
Receptor-mediated endocytosis
4) Movement of proteins
across an epithelial layer
(transcytosis):
- Immunoglobulin G
(IgG) secretion in milk;
uptake in the gut of the
newborn (passive
immunity).
- Immunoglobulin
transport across
epithelium of gut

Lysosomes in endocytosis:
Receptor-mediated endocytosis





















Luminal
membrane
Basal
membrane
Epithelial cell Intestinal
Lumen
Blood or
Interstitial
fluid
Tight junction
IgG
F
c
region
endosome
Fc receptor
Epithelial cell

Many of the events in receptor mediated
endocytosis are similar to vesicle transport in the
secretory pathway.

Specific receptors are clustered together at sites
on the plasma membrane by binding to the coat
protein clathrin.
The cytoplasmic portion of receptors provide sites/
regions that recognize and determine which receptors
to internalize
1
Endocytosis step # 1: Formation of coated pits
Coat is formed from clathrin.
Three heavy chains and 3
light chains are assembled
into a triskelion.
Triskelions are assembled
into a basket-like structure on
the cytoplasmic face of the
vesicle.
Adaptor proteins connect
the cytoplasmic side of
receptors to clathrin.
2
3
1
Endocytosis step # 2: Coat assembly continues
until vesicle is formed and released into cytoplasm
Clathrin coated vesicles
used for both receptor-
mediated endocytosis
and for vesicle transport
from TGN to lysosome.
The adaptor proteins for
TGN are different from
those for plasma
membrane.


- Low pH of the endosome
releases ligand (cargo) from
the plasma membrane
receptor.
- Ligand/endosomes moves on
to fuse with the lysosome.
- Some membrane receptors
recycle back to plasma
membrane.
3
4
Endocytosis steps # 3 and 4: Uncoating and fusion
of the vesicles with endosomes
Example: Cholesterol uptake

Cholesterol is carried with apo-B
protein as LDL particle.
LDL receptor internalizes LDL.
Familial hyper-cholesterolemia
leads to elevated blood
cholesterol:
Mutations to LDLR gene (encodes
the LDL receptor)
Mutations to apoB gene
Tf transferrin
TfR transferrin receptor
Example: Iron uptake
Iron is released from transferrin
in endosome
VACUOLE
Vacuoles: various maintenance functions
Vacuoles are large vesicles that have a variety of functions.
Some protists have contractile vacuoles that help to eliminate
water from the protist.
In plants, vacuoles may have digestive functions, contain pigments
and/or poisons (defensive).
2012 Pearson Education, Inc.
Contractile
vacuoles
Nucleus
Central vacuole
Chloroplast
Nucleus
Endomembrane: summary