The Endomembrane System

morphine

paclitaxel

(Taxol)

nootkatone

cis-1,4-

polyisoprene

artemisinin resveratrol

allicin

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

Ca2+ storage Ca2+-ATPase pumps Ca2+ into the lumen

Sarcoplasmic reticulum in skeletal muscles releases Ca2+ 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- (-NH2) 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 + Pi = G proteins

This energy is used to bind the ribosome to the translocon and release SRP.

BiP

3

2

GDP

+ Pi

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.

ER

GIC

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)

- Fe3+ 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

Fc 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