Alberts • Johnson • Lewis • Morgan • Raff • Roberts • Walter!
Molecular Biology
of the Cell
!
Sixth Edition
Chapter 12
Intracellular Compartments and
Protein Sorting
Hsou-min Li
[email protected]!
Copyright © Garland Science 2015
CHAPTER CONTENTS
The compartmentalization of cells
The endoplasmic reticulum
The transport of molecules between the nucleus
and the cytosol
The transport of proteins into mitochondria and
chloroplasts
Peroxisomes
THE COMPARTMENTALIZATION OF CELLS
All Eukaryotic Cells Have the Same Basic Set of
Membrane-enclosed Organelles
Plant cells have additional unique features: chloroplast
(plastid), cell wall and a very large vacuole
A typical animal cell is between 10 and 100 micro-meters.!
compartmentation <-> function!
Different cell types have very different abundance and shapes of organelles (membranes)
Liver: a lot more mitochondria!
pancreatic cells: a lot more secretory vesicles!
Why do we have the
problem of “protein
only one kind of !
cytosolic ribosome!
sorting”?
-- proteins are made at the
same place but have
to be sorted to
different organelles in
order to function
properly.
Generally divided into:
1. secretory pathway :
mostly cotranslational
2. organelle biogenesis:
mostly posttranslational
Common mechanism for
protein sorting to all
organelles:
1. A targeting sequence
2. A receptor (complex)
3. A translocation channel
across the membrane
4. An energy favorable
system to drive the
transport and make
the transport unidirectional.
Next !
chapter!
Organelle biogenesis!
So for all the systems we talk about today, we will ask 4 questions:
1. What is the nature of the targeting signal? (e.g. How does it different from other
signals?) What is the specific sequence motifs?
2. What is the receptor?
3. What is the structure of the channel? (e.g. are the proteins translocated in
folded or unfolded forms?)
4. What is the source of energy? ( i.e. why is the translocation uni-directional?)
ATP? GTP? PMF?
Proteins are guided to their destined compartment through specific pathways (previous slides). No
matter which pathway, they are all guided by sorting signals in their amino acid sequence that
function either as signal sequences or signal patches. Sorting signals are then recognized by
specific receptors.
(SKL)
(KDEL)
The Endoplasmic Reticulum: Introduction
pancreatic cell!
smooth tubules,
rough sheets!
ER is structurally and functionally diverse!
Steroid hormone secreting cells!
1. continuous with outer nuclear envelope!
2. Two basic types: rough (sheets), smooth (tubules)!
“Rough” example:!
Pancreatic cells need to secrete large amounts of digestive enzymes (proteins!) so they are loaded with
rough ER. !
“Smooth” example:!
Steroid hormone (lipids!) secreting cells have abundant smooth ER to house the lipid synthesizing and
modifying enzymes. !
ER network
History!
Rough and smooth ER can be isolated = microsomes
lighter fraction: mixtures of
smooth ER, Golgi.....!
heavier fraction: all from rough ER!
The isolated microsomes are powerful tools
for studying ER protein transport. For
example, from the experiments at left, they
found:!
1. Transport needs to be co-translational. !
2. proteins become smaller after being
transported into ER – a cleaved signal peptide.!
!
THE ENDOPLASMIC RETICULUM
•  Signal Sequences Were First Discovered in
Proteins Imported into the Rough ER
1971
Biomembranes 2 p193-195 (1971)!
1975!
The 1975 signal hypothesis (1999 Nobel Prize), Günter Blobel predicted:!
1. an N-terminal localized targeting signal on the protein itself.!
2. a binding factor that guides the protein to ER (deleted in the formal hypothesis, sigh!)!
3. a signal-induced protein-conducting tunnel through the ER membrane.!
The general process of co-translational import into ER
Then we talk about individual components in more details.!
Signal-Recognition Particle (SRP)
P54: !
binds the signal sequence,!
interact with SRP receptor,!
hydrolyze GTP !
blocking the elongation factor
binding site!
The GTPase switch
13!
Signal-Recognition Particle (SRP) and SRP receptor (SR) and GTP
Both SRP and SRP receptor are
homologous GTPases. They stimulate
each other’s GTP hydrolysis and then
release each other. Thus GTP ensures
the directionality of the SRP cycle.
GTP is also used by ribosome for
translation.!
polysome
Each mRNA is translated by many ribosomes at the same time- polysome (polyribosome). Ribosomes are all
the same. They form “free” polysome, or “membrane-bound” polysome, depending on which RNA they are
translating. !
Schematic representation of membrane-bound polysomes
together with microtubules that are linked to the ER membrane
via a microtubule-anchoring protein. Two ribosomes that are not
part of the polysomes are also shown.
The Polypeptide Chain Passes Through an Aqueous
Channel - the Sec61 complex
Identified by yeast mutants (that fail to secret) and confirmed by
cross-linking experiments. Composed of three proteins Sec61
α, β, γ, with Sec61α being the major component.!
!
How does the channel prevent "leaking"? : At "closed" state, the
channel is sealed by a plug (formed from one of the α helixes of
the Sec61α subunit) and an isoleucine ring at the neck of the
channel (a hydrophobic gasket). !
Channel is opened by the nascent chain, which inserts as a
loop with the signal peptide intercalated into the lateral
gate. !
Membrane proteins can diffuse laterally into the lipid bilayer
through the lateral gate of the channel.!
17!
history of the channel!
pore induced to form by signal peptide!
pore always open, sealed by ribosome!
pore usually closed by a plug!
pore opened by signal peptide!
The 1975 signal hypothesis predicted:!
1. an N-terminal localized targeting signal on the protein itself.!
2. a binding factor that guides the protein to ER (deleted in the
formal hypothesis, sigh!)!
3. a signal-induced protein-conducting tunnel through the ER
membrane.!
1971
Biomembranes 2 p193-195 (1971)!
1975!
The picture shows pores formed by 4 trimers
(α, β, γ). Cell Vol 87 (4), 1996 !
JCB 67, p835-851 (1975)!
18!
The role of ER and
Golgi in secretion.!
!
Discovery of
ribosome (“Palade
granules”), rough ER
and the internal
structure of
mitochondria.!
James Rothman!
Randy Schekman!
The “Signal Hypothesis”!
2013
The next Nobel prize on protein trafficking will be:!
Schekman and Rothman on vesicle budding and fusion.!
Translocation Across the ER Membrane Does Not Always Require Ongoing
Polypeptide Chain Elongation – post-translational transport into ER
Sec63 contains a J domain.
Post-translational translocation: Some proteins are fully translated before
being translocated across the ER membrane. Fairly common in yeast, and
occur occasionally in higher eukaryotes. !
Do NOT need: SRP , SR nor GTP. A direct interaction between the
translocon and the signal sequence appears to be sufficient for targeting to
the ER membrane.!
Need: Sec61 complex, Sec63 complex, Bip and ATP!
Membrane protein insertion into the ER membrane
Topology of all membrane proteins in the
secretory pathway is determined during
insertion into the ER membrane. When reaching
the plasma membrane, cytosolic side remains in
the cytosol and the lumenal side becomes the
extracellular side. !
*!
*!
*!
*!
"Inside" = do not cross membrane!
1. The positive-inside rule
+!
N positive!
SRP!
C positive!
SRP!
+!
Topology of an ER membrane protein
is determined mostly by three factors:!
!
1. “positive inside rule” for the first
transmembrane domain!
!
2. how many transmembrane domains
the protein have!
!
3. whether the first transmembrane
domain – usually the signal sequence –
is cleaved. !
2. Use hydropathy plot to predict the number of transmembrane domains!
+!
If you reverse the charge distribution on the two sides of the first transmembrane domain, the protein
topology will be flipped to become N-cytosol , C-lumen.!
3. Is the signal peptide cleaved?
stop transfer, start transfer?!!
-> during membrane protein
translation, transmembrane
domain (hydrophobic) will always
come and associate with the
Sec61 channel.!
ER import summery
ER Tail-anchored Proteins Are Integrated into the ER Membrane by a
Special Mechanism
Because even when translation is finished, the “signal peptide” (the hydrophobic transmembrane domain)
still has not emerged from the ribosome. When it emerges, ribosomes are gone. !
!
GET mutants: Golgi-ER transport!
e.g. SNARE proteins!
Translocated Polypeptide Chains Fold and Assemble in the Lumen of the Rough ER
Most proteins are fully folded and assembled in the ER. Factors that help folding:!
chaperone proteins (for example: BiP)!
S-S formation through protein disulfide isomerase!
glycosylations: N-linked (and O-linked)!
(some cytosolic and nuclear proteins are also gycosylated, but only by an single N-acetylglucosamine)!
N-linked: Most proteins synthesized in the rough ER are glycosylated by the addition of a common N-linked oligosaccharide.
A block of 14 sugars linked to the NH2 side chain of asparagine. Start to being trimmed immediately after addition.
Sec61&
One copy of this
oligosaccharyl
transferase sits with
each Sec61 translocon.!
Bound by calnexin and calreticulin!
Synthesis of the N-Linked oligosaccharide precursor
The N-linked Oligosaccharides Are Used as “handles” and “timers” for Protein Folding in the ER
They provide binding sites for calnexin and calreticulin to keep proteins from aggregating so other
chaperones can have time to help folding.!
The third glucose is removed then added continuously (like a timer). When proteins stay too long and still
can’t be folded, they are translocated out of the ER for degradation by the proteasome system in the
cytosol.!
One example:!
Misfolded soluble proteins
are recognized (see above),
bound by various proteins
(prevent aggregation so they
can be translocated out, also
to reduce the S-S bond),
targeted to a translocator,
extracted by an ATPase in
the cytosol,
polyubiquitinated, deglucosylated and degraded
by the proteasome.
Misfolded membrane
proteins follow a similar
pathway but use a different
translocator.!
O-linked: to -OH groups of serine and threonine. One-by-one added from sugar nucleotides in ER and Golgi.!
Golgi.!
ER !
ABO blood types (O-linked
glycan attached to glycoproteins
and glycolipids on the surface of
erythrocytes). The presence of
absence of glycosyltransferase that
add N-acetylgalactosamine or
galactose to O-antigen determines
a person’s blood type.!
31!
Misfolded Proteins in the ER Activate an Unfolded Protein Response
Three examples of known mechanisms: they produce different transcription factors that go into the
nucleus to activate the transcription of genes encoding proteins that will assist folding
PERK
phosphorylates
initiation factor –
inhibit translation
of most proteins,
but some proteins
(e.g. transcription
factors required
for unfolded
protein response)
are preferentially
translated.
IRE1
(XBP1/Hac1)!
ATF6
UPR signalling mediated by IRE1 and ATF6.
Zhang K , Kaufman R J J. Biol. Chem. 2004;279:25935-25938
Some Membrane Proteins Acquire a Covalently Attached Glycosylphosphatidylinositol (GPI) Anchor
in the ER
Functions of GPI anchor:!
1. faster diffusion on the plasma
membrane!
2. collecting in the lipid raft!
3. serving as a signal for polarized
transport (next chapter).!
THE TRANSPORT OF MOLECULES BETWEEN THE NUCLEUS AND
THE CYTOSOL
The outer nuclear membrane is continuous with the ER membrane
(both have ribosome attached) and the perinuclear space is continuous
with the ER lumen, with similar protein composition. !
!
The inner nuclear membrane has distinct protein composition, with
lamina the most well known one. Lamina act as anchoring for
chromosomes and cytoplasmic cytoskeleton via protein complexes
that span the nuclear envelope. !
!
Nucleus has large “nuclear pores”.!
!
Nuclear Pore Complexes Perforate the Nuclear Envelope
look in from cytosol
Nuclear pores are built with different “nucleaoporin” proteins (ring,
scaffold, channel....). The disordered region of channel nucleoporins
forms a “kelp bed” like mesh in the pore to allow diffusion of small
molecules but restrict large molecules, and also to provide docking sites
for nuclear import receptors (see below). Even without the help of the
nuclear transport machinery, proteins less than 60 kD can diffuse through
by themselves. With the machinery, partially assembled ribosomes can
be ferried through. !
look out from inside the nucleus
Nuclear Localization Signals Direct Nuclear Proteins to the Nucleus
Nuclear localization signals (NLS) are usually short stretches of positively charged amino acids. They can be anywhere
in the proteins and are not processed after reaching inside the nucleus. Proteins without a NLS can bind to another
protein with NLS and be transported. NLS can even be coated onto gold particles and result in transport.!
Gold particles
coated with NLS
peptides, injected
into cells and
fixed after various
amount of time.!
immunofluorescence microscopy!
Nuclear Import Receptors Bind to NLS
Nuclear import receptors (and export receptors) are a
family of homologous proteins but each family member
has slightly different binding sequence specificities. !
Proteins without NLS (“cargo 4” in the figure) can
bind to adaptors with NLS and then be imported.!
It is often difficult to tell from receptor sequences
whether they are import of export receptors. Export
signals are much less conserved and less understood. !
The GTPase switch
39!
Nuclear Import: The Ran GTPase Imposes Directionality on Transport
Through NPCs
Ran-GAP is only in cytosol – high
concentration of Ran-GDP in the
cytosol!
Ran-GEF is only in nucleus- high
concentration of Ran-GTP in the
nucleus.!
1. Nuclear Import Receptors Bind to
Nuclear Localization Signals on
the cargo (or just empty receptor)
and then bind to the phenylalanineglycine (FG) repeats of the
unstructured domains of the
channel nucleoporins.!
Import driven by cargo complex
concentration gradient.!
Ran-GTP binds to receptor in the
nucleus and causes cargo release.
Ran-GTP-receptor travel back to
cytosol due to concentration
gradient. Ran-GTP converted to
Ran-GDP (Ran-GAP is only in the
cytosol).!
2. Ran-GDP does not like to bind to
import nor export receptor so
receptor is released.!
Import!
high Ran-GDP
concentration
high Ran-GTP
concentration
Nuclear Export Works Like Nuclear Import, But in Reverse
Ran-GAP is only in cytosol – high
concentration of Ran-GDP in the
cytosol!
Ran-GEF is only in nucleus- high
concentration of Ran-GTP in the
nucleus.!
1. Ran-GTP binds to receptor in the
nucleus and promotes cargo binding.
Export driven by Exportin-Ran
gradient. Ran-GTP converted to
Ran-GDP.!
2. Ran-GDP does not like to bind to
import nor export receptor so
receptor is released.!
3. Empty export receptor binds to
nucleoporin and enter the nucleus
again. !
Export!
Controlled shuttling of proteins in and out of the nucleus by controlling the exposure of
NLS (nuclear localization signal) and NES (nuclear export signal)
Ca2+ concentration and phosphorylation control whether
the NLS and NES are exposed: When T cells are
activated, intracellular Ca2+ concentration rises. In high
Ca2+, calcineurin binds to NF-AT (nuclear factor of
activated T cells) and dephosphorylates it, resulting in
exposure of NLS and blocking of NES. NF-ATcalcineurin complex is imported into the nucleus. When
the activation shuts off and Ca2+ drops, calcineurin
releases NF-AT, resulting in exposure of NES and rephosphorylation of NLS.!
Some of the most potent immunosupressive
drugs inhibit the ability of calcineurin to
dephosporylate NF-AT.!
THE TRANSPORT OF PROTEINS INTO MITOCHONDRIA AND
CHLOROPLASTS- Introduction
1. Double-membraned envelope;!
!many sub-compartments;!
!proteins need to cross two
membranes!
2. with their own DNA
(transcription and translation
machinery)!
lumen
Mitochondria
Mitochondria typically form extended networks
throughout the cytosol, and their overall morphology is
controlled by organelle fusion and fission. The
ultrastructural organization of mitochondrial membranes
varies considerably between mitochondria of different
organisms, tissues, and cell types. The diversity of
mitochondrial architecture reflecting the diversity of their
metabolic functions is apparent when comparing
mitochondria from pancreas (A), adrenal cortex (B),
astrocytes (C), muscle (D), brown adipose tissue (E), and
an Amoeba (F). Not surprisingly, aberrant mitochondrial
shapes and ultrastructures are observed in pathological
situations. Cell 149, April 27, 2012 ©2012 Elsevier Inc.
DOI 10.1016/j.cell.2012.04.010!
Mitochondria Signal Sequences
Amphiphilic alpha helix !
one side hydrophobic: interact with the receptor
on the outer membrane!
one side positive charged: for crossing the inner
membrane !
budding yeast. !
green: mitochondria.!
blue: bud scars!
positive charged side!
!
hydrophobic side!
!
Basic steps into matrix
proteins transported in
unfolded forms!
!
hsp70!
• 
• 
• 
TO(I)M= translocon of the outer (inner) membrane of mitochondria
proteins are imported post-translationally
Mitochondrial Precursor Proteins Are Imported as Unfolded Polypeptide
Chains
• 
imported through contact sites where two membranes are pressed
together
• 
energy:
ATP hydrolysis by Hsp70, in the cytosol and in the matrix
membrane potential (the electrical components of the H+ gradient
across the inner membrane). Matrix side negative so positivecharged signals can cross.
Mitochondrial Protein Translocators:
more than TOM and TIM
Mitochondria have specialized
complexes for assembly proteins
into various subcompartments.
!Some of the complexes
are newly evolved. Some are from
bacterial. !
SAM and OXA complexes are
evolved from bacteria!
Bacteria and mitochondria use similar mechanisms to insert outer-membrane
beta-barrel proteins into their outer membrane: the SAM and BAM complexes
SAM: The sorting and assembly machinery
BAM: beta-barrel assembly machinery
bacteria
eukaryotic cell mitochondria
Transport Into the Inner Mitochondrial Membrane and Intermembrane Space Occurs Via
Several Routes
inner membrane!
intermembrane space!
TRANSPORT OF PROTEINS INTO CHLOROPLASTS
Signal: rich in serine, usually about 50-60 amino acids, longest among organelles.!
TO(I)C=translocon at the outer (inner) envelope membrane of chloroplasts!
1. Proteins also post-translationally imported. Similar to mitochondria in the need of crossing two membranes, but whether
proteins transported in unfolded or folded forms, not clear yet. !
2. Use ATP and GTP (the two receptors, Toc159 and Toc34, are GTPases), not membrane potential!
Hsp90
Hsp90
3. Two signal sequences direct proteins to the thylakoid membrane and lumen in chloroplasts!
4. At least three pathways for import into thylakoid – all derived from bacteria!
Two for thylakoid lumen:
(1) ΔpH (Tat) for folded
protein, use ΔpH
(2) SecA for unfolded protein
(e.g. plastocyanin), use high
ATP and SecY (Sec61
homolog)
At least one for the thylakoid
membrane: SRP/Alb3: for
LHCP
E. coli has homologous pathways!
!
One difference:
SRP/SR for cotranslational
insertion of very
hydrophobic
membrane
proteins into the
inner
membrane.!
Peroxisome: Introduction
1. Single membrane. No DNA
2. Peroxisomes use molecular oxygen and hydrogen
peroxide to perform oxidation reactions (marker
enzymes: catalase, urate oxidase. There is so much of
them that they form crystals).
RH2+O2 ! R+H2O2
H2O2 + R’H2! R’+2H2O e.g. oxidation of the ethanol we
drink
2H2O2 ! 2H2O + O2 (catalase)
3. Another major function: breaking down fatty acids
through beta oxidation to make acetyl CoA
4. animal peroxisome: synthesize plasmalogen (the first
two steps are in peroxisome) – the most abundant
phospholipids in myelin – why peroxisomal disorders
lead to neurological disease
5. Plant peroxisome – a subclass called glyoxisome, which
can take two acetyl CoA to make one succinic acid –
turn fatty acids into carbohydrates.
E. Newcomb!
Peroxisomes proliferate through two mechanisms: de novo biogenesis through
precursor vesicles budding off from ER, and fission of pre-existing peroxisome
Pex19
required for
vesicle
budding from
ER!
1!
2!
3!
1. some membrane proteins insert
into ER membranes directly. Some
of these serve as receptors for
matrix protein import. !
2. PTS1 and PTS2 pathways
converge at the membrane channel.!
3. Peroxisomes import proteins in
folded forms (gold particles coated
with PTS1 can be imported).
Receptor recycling requires monoubiquitination and ATP.!
4. Receptor poly-ubiquitination
results in receptor degradation. !
4!