Intracellular Compartments

and Protein Sorting

Reading: Alberts et al. 5th edition
695-707, 713-716, 721-727, 766-769, 779-783

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 Intracellular Compartments

Cytosol - half the cell volume

- protein synthesis and degradation occurs here
- intermediary metabolism (degradation and synthesis of small molecules)
not dna, rna; like glucose and others

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Table 12-1 Molecular Biology of the Cell (© Garland Science 2008)
 Definition: organelle
A subcellular compartment or large macromolecular
complex, often membrane-enclosed, that has a
distinct structure, composition, and function.
Examples of membrane-enclosed organelles:
- Nucleus
- Endoplasmic Reticulum
- Golgi Apparatus

Examples organelles that are not membrane-bound:

- Nucleolus
- Centrosomes
made of two centrioles at 90' angles

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 not an organelle
(endocytosis)
(Digestive Enzymes)
(modifies proteins & lipids)
(oxidative (ATP synthesis)
reactions)
(ER)

(protein synthesis) (RNA, DNA synthesis)

rough endoplasmic reticulum

RER – synthesis of transmembrane, organellar and secreted proteins

SER – phospholipid synthesis, detoxification
smooth endoplasmic reticulum

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Figure 12-1 Molecular Biology of the Cell (© Garland Science 2008)
 the amount of membrane
surrounding every
organelle in the cell is
greater than the plasma
membrane

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Figure 12-2 Molecular Biology of the Cell (© Garland Science 2008)
 around half around half (RER &
SER)
RER is for secreted
have more SER because liver detoxifies you proteins, exocrine cells
secrete digestive
enzymes, (RER
excretes)

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Table 12-2 Molecular Biology of the Cell (© Garland Science 2008)
 Intracellular Compartments
are Dynamic
They exchange components:
 lipids blue arrows - proteins and lipids move intracellularly

 proteins

-exports/secretes
-branches from
nucleus

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Figure 12-5 Molecular Biology of the Cell (© Garland Science 2008)
 The Endomembrane system the red enclosed parts; they all help each other
and work together
- the vacuoles in plants also a part of this system

/vacuole (in plants)

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Figure 12-1 Molecular Biology of the Cell (© Garland Science 2008)
 Endomembrane system is involved in BOTH of these pathways!!

A) Biosynthetic/Secretory
pathways
– Biosynthesis
• Proteins and lipids made
in the ER delivered to the
membranes of other
organelles
– Secretion
• secreted proteins move OUT of the cell

B) Endocytic Pathway
• endocytosis - material moving INTO the cell

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 secretion to outside

basically the reverse

consumption from
outside

A) exocytosis: vesicle contents  are delivered to the extracellular space

vesicle membrane  become part of the plasma membrane

B) Endocytosis: PM (plasma membrane)  forms the vesicle membrane

vesical luminal
contents come from  extracellular space

What about the individual leaflets of the membranes?

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Figure 13-1 Molecular Biology of the Cell (© Garland Science 2008)
 Vesicular Transport
Vesicle: small, membrane-enclosed
organelle in the cytoplasm of a
donor compartment

one giving something

eukaryotic cell

They shuttle components back and

forth in the endomembrane system

For example:
from the ER to the Golgi target compartment

destination of material
transport
Luminal leaflet
*The leaflets' sides do NOT
change!*

Cytosolic leaflet
blue faces the cytosol

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Figure 12-7 Molecular Biology of the Cell (© Garland Science 2008)
 How are specific proteins targeted to
different organelles?
 mRNA arrives in the cytoplasm and translation
starts on ribosomes in the cytosol.
 A cytosolic protein: translated in the cytosol will have no sorting schematic

http://bcs.whfreeman.com/lodish5e/
Select Chapter 16
Moving Proteins into Membranes and
Organelles
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 Sorting Proteins to Mitochondria & Chloroplast

Mitochondria and chloroplasts have their

own genome and ribosomes BUT most
proteins are nuclear-encoded
– translated in cytosol
– targeted by a signal sequence Page 713 Molecular Biology of the Cell 5/e

– imported into the organelle

 sorting is post-translational
proteins remain
unfolded in cytosol by
association with hsp70
chaperones
hsp70 keeps proteins from folding

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 Lets follow the path of a secreted protein
from translation to secretion. Ribosome
mRNA
• mRNA arrives in the
cytoplasm and translation
starts on ribosomes in the Signal sequence
amino acid
cytosol.

• While translation is still

cytosol
occurring insertion of the
protein into the ER starts ER Lumen

cotranslational translocation (all at the same time)

inserted in ER AS it is made

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Figure 12-35a Molecular Biology of the Cell (© Garland Science 2008)
 signal sequence says "Take entire thing,
ribosome, AAs, etc. to the ER"

protein to be secreted helps protein cross

into the lumen

cleaves off signal peptide that tells AA chain to

go to ER

ER

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Figure 12-38 Molecular Biology of the Cell (© Garland Science 2008)
Pancreatic cells make lots of secreted
proteins
• Use these cells to follow the
path of newly synthesized
proteins.
– Provide the cells with a short
“pulse” of radioactive amino
acids (AA).
– Follow the path of these AA
as they are incorporated into
proteins.

A Technique called a:
 pulse-chase experiment
give cells radioactive AAs (pulse)
follow where they go (chase)

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Proteins move from the...
RER lumen to the Golgi apparatus then to secretory vesicles from where they leave the cell.

(chase time) (chase time) (chase time)

Red dots indicate the location of the labeled proteins 17

Secretory Pathway

• Constitutive Pathway:
– Continual production of
secreted proteins.
– e.g. collagen
Collagen

• Regulated Pathway:
– Proteins are stored in
secretory granules ready
secretory granules

for export in response to

a stimulus.
– e.g. Neurotransmitter
Neurotranscripts (transmitters?)

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 Protein Sorting Mechanisms
(both ways; only nucleus to cytosol and v.v.)

1. Gated: proteins move between

the cytosol and nucleus
through NPCs
- Nuclear pore complex

2. Transmembrane: (all one-way)

protein translocators needed to
Protein translocators

transport specific proteins

across a membrane.

3. Vesicular: membrane-enclosed
transport vesicles ferry proteins
from one compartment to
another.
KNOW THIS~!
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Figure 12-6 Molecular Biology of the Cell (© Garland Science 2008)
 Signal Sequences
• A stretch of the amino acid sequence of a protein
that directs the protein to the correct location in
the cell.
• Each signal sequence specifies a specific
destination in the cell.
 Nucleus, cytosol, mitochondria, peroxisomes, ER, etc.

• Signal sequences are recognized by sorting

receptors that take proteins to their destination.
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 Protein Sorting to the
Endoplasmic Reticulum

• http://www.dnatube.com/video/544/Signal-
Recognition-Particle-SRP

The protein shown ends up in the lumen of the

endoplasmic reticulum.
signal recognition particle brings ribosome to the ER

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 Signal Sequences
• Often found at the N-
terminus of the protein.
• Signal peptidases: can
Signal peptidase
NH2
remove the signal sequence
from the finished protein.

• Can be internal stretches of

AA which remain part of the
protein. Not cut away, can be 6 to 136 AAs long

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Recap: Sorting of a Secreted Protein.
• Translation starts on cytosolic ribosomes.
• Signal sequence at the amino-terminal end directs the
protein to the ER.
• The signal sequence is: hydrophobic
hydrophobic; cotranslational translocation

• This is: COTRANSLATIONAL

Co-translational translocation to ER
TRANSLOCATION

• Protein inserted through the membrane by a:

– protein translocator.
protein translocator

• Signal sequence cleaved, left behind in the ER **only if an N-terminus

sequence
membrane, secreted protein ends up in the ER lumen.
• Secreted protein moves in transport vesicles via the
secretory pathway.
• Released by  exocytosisExocytosis at the PM.
*GOES TO GOLGI BEFORE GOING OUTSIDE OF THE CELL
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 What about a transmembrane protein?
transmembrane protein

• CytosolER Ribosome here somewhere

SRP sticks
here

degraded

hydrophobic

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Figure 12-46 Molecular Biology of the Cell (© Garland Science 2008)
 Two different ways a single pass transmembrane
protein can be oriented in the ER membrane
(for a protein with an +ve side faces the cytosol
internal signal sequence)
not on N-terminus

-ve faces ER lumen

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Figure 12-47 Molecular Biology of the Cell (© Garland Science 2008)
 A Multipass Transmembrane Protein

+ +

_ _

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Figure 12-48 Molecular Biology of the Cell (© Garland Science 2008)
Lets follow the path of a transmembrane protein
from translation to the plasma membrane (PM).
1. CytosolER

2. ER Golgi

What’s going on
in the Golgi?
glycosylation
- adding sugars, lipids, and
proteins

3. Golgi  PM
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 Definition
• The Golgi apparatus receives proteins and
lipids from the endoplasmic reticulum,
modifies them, and then dispatches them to
other destinations in the cell.

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 The Golgi Apparatus
Nuclear envelope

ER

faces ER

faces plasma membrane 29

Figure 13-25a Molecular Biology of the Cell (© Garland Science 2008)
 Protein Glycosylation
• Actually starts in the ER: A single type of oligosaccharide is
attached to many proteins.
• Complex oligosaccharide processing required for
mature proteins occurs in the Golgi

Golgi:
multi-stage processing unit, with
different enzymes in each cisterna

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Figure 10-28b Molecular Biology of the Cell (© Garland Science 2008)
Maintenance of membrane
protein asymmetry

• Each protein is inserted

into the membrane in
the ER in a specific
manner
only modified on the lumenal side

result of membrane insertion process

• This protein asymmetry

is maintained through
vesicular transport
lumenal side will be on exterior;
cytosolic will stay cytosolic *Not due to the nature of the protein, simply the way they were inserted;
if mature proteins are isolated and reconstituted into another membrane,31they end
up in either orientation. Go LOOK AT DETERGENTS*** lecture 4
Recap: Sorting of a PM protein.
• Translation starts on cytosolic ribosomes.
• Signal sequence directs the protein to the ER. Signal recognition protein
• There are different ways the protein can be inserted
into the membrane.
• The signal sequence is: hydrophobic
• This is: Cotranslational
Co-translational
translocation!!!
translocation to ER
• Protein inserted through the membrane by a:
– protein translocator.
protein translocator

• Protein moves in transport vesicles to the PM

• Vesicle membrane fuses with the PM during
Exocytosis, transferring the protein to the PM
 Exocytosis
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 Lysosomes and Endosomes
proteins can go forward or backwards; cell doesn't only export stuff

golgi vessicles fuse

vessicle from the exterior

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Figure 13-3b Molecular Biology of the Cell (© Garland Science 2008)
 Definitions
• Endosome: membrane-bound organelle that
carries materials newly ingested by endocytosis
and passes them to lysosomes for degradation.

• Lysosome: a small sac of digestive enzymes that

functions in degrading worn-out organelles, as
well as macromolecules and particles taken into
the cell by endocytosis.
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 Endocytosis
Endocytosed material found in the:
Early endosome

A leukocyte ingesting a yeast cell

Lysosomal proteins are
delivered in vesicles from
the Golgi




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 Lysosomal proteins are found in
heterogeneous looking compartments

Lysosomal proteins delivered to endosomes directly from the Golgi in vessicles

Mature lysomes fuse with late endosomes to recycle material

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Figure 13-38 Molecular Biology of the Cell (© Garland Science 2008)
 Lysosomes are the main site of
intracellular digestion
• Contain approx 40 types
of hydrolytic enzymes:
– Proteases, nucleases,
lipases etc.
• These enzymes are:
– Acid hydrolases
– Require an acidic
environment proteins don't work in high pH
• Uses an ATPase to pump H+ ions into
the lumen, creates acidic environment

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Figure 13-36 Molecular Biology of the Cell (© Garland Science 2008)
 Lysosomes
• Low pH requirement of lysosomal
enzymes protects contents of the
cytosol from digestion.
• Lysosomal membrane proteins are
glycosylated to protect them from
proteases in the lumen
• Transport proteins in the lysosomal
membrane transfer the products of
digestion out of the lysosome to the
cytosol
 Ex. amino acids, sugars

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Figure 13-37 Molecular Biology of the Cell (© Garland Science 2008)
 Formation of lysosomes
• Lysosomal proteins from the ER and Golgi
are incorporated into endosomes at different stages.
• “classical” lysosomes:
– Majority of endocytosed
material has been digested

However lysosomal
hydrolases delivered
from the Golgi are found in
 early/late endosomes
Where do lysosomal membrane lipids come from? The golgi and plasma membrane
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Figure 13-3b Molecular Biology of the Cell (© Garland Science 2008)
 Plant Vacuoles
large, fluid-filled vessicles
• Occupy 30-90% of the cell volume
• Diverse
• Involved in:
– Digestion
– Nutrient storage
– Waste storage
– Increasing cell size
– Turgor pressure
maintaining osmotic balance ; pushes against cell wall to prevent wilting

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Figure 13-39 Molecular Biology of the Cell (© Garland Science 2008)
 A large increase in
volume of a plant cell
without increasing
the volume of the
cytosol

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Figure 13-40 Molecular Biology of the Cell (© Garland Science 2008)
 Peroxisomes
• Use molecular oxygen to
oxidise organic molecules
• Oxidative reactions
produce hydrogen
peroxide (H2O2)
• H2O2 is toxic; also
degraded in peroxisomes
• Function in the
breakdown of long fatty
acid chains.
in liver and kidney cells to help degrade alcohol

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Figure 12-30 Molecular Biology of the Cell (© Garland Science 2008)
 Model of Peroxisome Formation

- Proteins inserted into the ER Found in

- Proteins imported from the cytosol peroxisomes
transmembrane insertion again, w/ start and stop transfer sequences
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Figure 12-33 Molecular Biology of the Cell (© Garland Science 2008)
 Overview: Protein Sorting (1)
• movement of proteins
 within cell between
different compartments
 out of cell
 into cell
can all occur
• protein synthesis initiated on
ribosomes in cytosol

• proteins must be sorted to the correct

location signal sequences
Cytosolic proteins stay
stay in thecytosol
in the cytosol, nosignal
w/ no signal sequence
sequences
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 Overview: Protein Sorting (2)
1) Post-translational process
 in some organelles, can be cotranslational
proteins made in the cytosol, and are
sometimes unfolded
• Unfolded: mitochondria, plastids Nucleus
• Folded: nucleus, peroxisomes

mRNA
2) Co-translational process
ribosome
• Endoplasmic Reticulum (ER)
• proteins with ER Signal Sequence
 Nuclear localisation signal (NLS); proteins protein ER
synthesis membrane
are already folded

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 How does a nuclear protein get into
the nucleus?
• Transport is:
 gated; nuclear pore complex (NPCs)

• Occurs through:
 Nuclear pore complex (NPCs)

This process can be regulated

by intracellular signals.
e.g.
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Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008)
 Protein Sorting: Gated Transport

• Proteins moving between cytosol and nucleus

• Nuclear Pore Complex:

 selective transport
of macromolecules

free diffusion of

small molecules
(<5,000 daltons)

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Figure 12-10 Molecular Biology of the Cell (© Garland Science 2008)
 Nuclear Import Signal Sequences

Nuclear import signal sequences: with a mutation, these proteins will stay
in the cytosol

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Figure 12-11 Molecular Biology of the Cell (© Garland Science 2008)
Remember....

activate transcription increase frequency of transcription

nuclear receptors can function as

activators for transcription;
estrogen is an activator

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The Estrogen Receptor (ER) is a ligand-
modulated regulator of transcription
• When estradiol is NOT present
 estrogen receptor is in the cytosol, NLS is hidden
• When estradiol enters the cell
 NLS exposed, movement to the nucleus
can exposed the proteins (needed to go to the nucleus)
NLS

goes to nucleus and then starts transcription;

too much estrogen receptor can cause breast cancer

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 Protein Sorting: Mechanisms
gated transport transmembrane
transport

• requires protein
translocators

vesicular transport
• vesicles move protein
between compartments

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What gives the vesicles directionality?
• Directed movement of transport vesicles,
pulled by motor proteins associated with the
cytoskeleton

• A movie to review protein sorting:

http://www.youtube.com/watch?v=PUy_Em5dXmc

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End

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