Molecular Cell Biology

Fifth Edition

Chapter 16:
Moving Proteins into Membranes
and Organelles
So far, two fundamentally different ways of
translocating proteins are known: Chap. 16: proteins
are delivered in soluble forms. Chap. 17: proteins are
delivered in vesicles.

Harvey Lodish • Arnold Berk • Paul Matsudaira •

Chris A. Kaiser • Monty Krieger • Matthew P. Scott •
Lawrence Zipursky • James Darnell

Hsou-min Li 2006
1

Why do we have the

problem of “protein
sorting”? only one kind of
cytosolic ribosome
-- proteins are made at the
same place but have
to be sent to different
organelles.

Generally divided into:

1. secretory pathway :co-
translational
2. organelle biogenesis:
post-translational

Common mechanism for

protein sorting to all
organelles:
1. A targeting sequence
2. A receptor (complex)
3. A translocation channel Organelle biogenesis
across the membrane
4. An energy favorable
system to drive the
transport and make
the transport
unidirectional So for all the systems we talk about today, we will ask 4 questions:
1. What is the nature of the targeting signal?
2. What is the receptor?
3. What is the structure of the channel? 2
4. What is the source of energy?
16.1 Translocation of secretory protein across the ER membrane

We will discuss:
1. signal peptide
2. SRP
3. SR and the GTP cycle
4. Sec61p channel
5. Post-translational translocation 3
6. Membrane protein topology

But let’s first talk about some old history: How did we know secretory proteins don’t just directly cross
the plasma membrane from the cytosol?

In addition: Import into ER has to be co-translational.

Because: Secretory proteins are localized in the ER lumen
shortly after synthesis

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How was the pathway defined?
a. Gorge Palade: pulse-chase with pancreatic cells (by injecting guinea pigs with [3H]leucine)
1974 Nobel Prize of Physiology and Medicine
(b. genetic evidence and c. glycan processing in Chapter 17)

3 min
after chase: 7 min:
ER Golgi

37 min: 117 min:

immature mature
secretory secretory
granules granules
5

1. ER
signal peptide

Mutations that disrupt the hydrophobic core disrupt translocation

Many artificial sequences containing a hydrophobic core can function as an ER signal sequence6
 2. SRP (signal recognition particle)
identification: high-salt washed ER membranes no longer function
in transport
--add back the wash solution and the activity was restored.
One RNA, 6 proteins. Different domains have different functions

3. SR (SRP receptor) and the GTP cycle

SR identified by protease treatment of ER membrane.

Reconstitution: only need ribosome/nascent chain complex, SRP, SR and the

Sec61p complex and GTP.
Therefore the only energy required is GTP, for SR/SRP and for translation
(“pushing”).

membrane
bound

4. Sec61p channel
Identified by yeast mutants (that fail to secret) and
confirmed by cross-linking experiments.
Composed of three proteins Sec61 !, ", #, with
Sec61! being the major component.

How does the channel open and close? Still

controversial, at least three hypotheses
1. Always open, sealed by Bip and ribosome
2. Pore formed by assembling 4 complexes upon
translocation
3. Diaphragm

1. Always open, sealed by Bip and ribosome

JCB vol 156, p218, 2002

8
 2. Pore formed by assembling 4 complexes upon translocation

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 (!, ", #).
Cell Vol 87 number 4, 1996

JCB 67, p835-851 (1975) 9

3. Diaphragm 2004 channel crystal structure: a plug formed by one of the ! helixes in the sec61! subunit. The plug
moves away when signal peptide binds.

!"#$%&'"()*(+,-$./*%(+-0"#++%1
!"#$%&'()*+,&*)$&!",)&-.&/0)1
2%314-5#6%-$./*%(+'-#.%-5/7%6-#0./''-0%1181#.
5%59.#+%'-*"./8,"-#-$./*%(+-0"#++%1:-;"%-0.4'*#1
'*.80*8.%-/)-*"('-0"#++%1-('-+/3-.%7%#1%6-#+6
0/+)(.5'-%<$%0*#*(/+'-*"#*-(*-58'*-0"#+,%-'"#$%-*/
#11/3-$./*%(+'-*/-$#'':

The isoleucine ring in the center of the hour-glass shaped

channel is too small for polypeptide chain to pass.
It is predicted that the backbones of the channel
have to move like diaphragm to increase the pore
size. 10
2=;>?@-ABC-DEF--G-H=2>=?I-EJJD-$ED&EKL-$MK&DD
 Nature vol 427 p 36-44 2004

The minimum unit is one copy of the heterotrimer.

10 !-helix trans-membrane domains of Sec61! form the
main body of the channel.
One of the alpha helixes of the alpha subunit forms the plug.
There is a hydrophobic pore ring in the center of the channel.
Membrane proteins can diffuse laterally into the lipid bilayer.
The pore seen in the previous EM was probably space among
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several actual channels.

5. Post-translational translocation: Some proteins are fully translated before being translocated across the
ER membrane. Fairly common in yeast, occur occasionally in higher eukaryotes.
Do NOT need: SRP , SR nor GTP.
Need: Sec61 complex, Sec63 complex, Bip and ATP

Polypeptide slides inward
and outward (Brownian
motion).
“Brownian
ratchet”
Bip binding prevents
Interaction of Bip-ATP backward sliding of the
with Sec63 (containing a J polypeptide.
domain) causes ATP
hydrolysis by Bip.
Bip-ADP binds stably to
unfolded polypeptides.

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16.2 Insertion of proteins into the ER membrane
Topology: number of times a membrane protein spans the membrane
and the orientations of the membrane spanning segments

Topology of all membrane proteins in the secretory pathway is

determined during insertion into ER.

Importance: proper function depends on correct topology

Q: which side will be intracellular, which side will be extracellular?

Topology is mainly determined by the

direction of insertion of the first
transmembrane domain (usually the
signal peptide), and whether the signal
is cleaved. The rest of the
transmembrane domains insert
accordingly. You can flip the entire
orientation by changing the charges
flanking the first transmembrane
domain.

The “positive inside” rule

13

N
N N
+N + + +

N
N N
N

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Topology prediction from sequence

1. To predict number of transmembrane domains: Hydropathy

profile (hydropathy plot) first assign a value of hydropathic
index to each amino acid then calculate the sum of 20 (of some
other number) consecutive amino acids along the entire length
of the polypeptide.
To predict orientation: positive inside rule

2. Sequence homology to other knows proteins

GPI (glycosylphosphatidylinositol) anchored proteins

This end won’t be interacting with
other cytosolic proteins or
cytoskeleton, so can diffuse more
freely. Why are all GPI-anchored
proteins extracellular surface proteins?

Another function: apical sorting: GPI anchor targets the attached

protein to the apical domainz of the plasma membrane in certain
polarized epithelial cells.
Synthesized GPI transamidase cleaves
as Type I ER the membrane anchor and
membrane 15
transfer the soluble
protein domain to preformed GPI

16.3 Protein modifications, folding, and quality control in the ER

Types of modification:
Glycosylation (addition and trimming)
proteolytic cleavage (processing) In both ER and Golgi
S-S formation
Folding Only in ER
Multi-subunit assembly
I. Glycosylation: mostly in ER and Golgi. Most Golgi resident proteins are glycosyltransferases. Very few cytosolic
proteins are glycosylated (a few transcription factors are). Recently identified: a transport pathway from cis Golgi to
chloroplasts and some chloroplasts proteins are glycosylated. By vesicles? Or soluble proteins? Or something totally
different?
O-linked: to -OH groups of serine and
Two types, N-inked and O-linked
threonine. One-by-one added from sugar
nucleotides in ER, cis-Golgi and trans-
Golgi. trans-Golgi.
ER, cis-Golgi
N-linked: to amide nitrogen of asparagine in the sequence Asn-x-Thr
or Asn-x-Ser. Synthesized also from sugar nucleotides, added in ER
first as a pre-assembled big block of 14 sugars
then diversely modified in ER and Golgi.

sugar nucleotide
precursors

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 Functions of the glycan groups
1. Promote proper folding (through calnexin, see
next page): mutation or treatment with
tunicamycin to inhibit glycosylation can
cause misfolding of some proteins
2. Promote stability in blood of many secreted
glycoproteins
3. Cell-cell adhesion: e.g. glycan on CAM (cell
adhesion molecules) of leukocytes binds to
sugar binding domains of CAM of
endothelial cells lining blood vessels.
4. Major antigens: ABO blood types (O-linked
glycan attached to glycoproteins and
glycolipids on the surface of erythrocytes.

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II. Disulfide bond formation: by Ero1,

protein disulfide isomerase (PDI) and
unknown oxidant.

Only in ER lumen and bacterial periplasmic

space. So only secreted proteins or
extracelluar domain of membrane
proteins have S-S.

E. coli over-expressed proteins often

aggregate due to lack of S-S formation
during synthesis

S-S is often re-arranged in ER lumen by PDI

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III: proper folding and multimeric protein
assembly (in ER):
Proteins that assist folding in ER
Hsp70 class chaperone: e.g. Bip
PDI!S-S
calnexin and calreticulin: bind to glycans thus prevent
folding of adjacent amino acids so PDI and
glycosyltransferases and other chaperones have
time to properly fold the polypeptide.
Peptidyl isomerase: accelerate the rotation about a
peptide bond (cis <--> trans)

A good example that contains all the modifications:

hemaglutinin (the spikes protruding from the surface of an Interaction of the membrane spanning !-helix from three
influenza virus) assembly subunits triggers the formation of a long stem containing
one ! -helix from the luminal part of each subunit.

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In the Golgi, more glycan processing Some proteins undergo proteolytic processing
after leaving the TGN: e.g. the processing to pro-
insulin to insulin takes place in the secretory
granules.

Why don’t secretory proteins just directly cross the plasma membrane from the cytosol?-- they have to go
through the processing/modification factory of ER and Golgi to make sure they are properly modified and
folded.

What if they can’t be properly folded ??

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 IV. The unfolded-protein response (Figure 16-22):
Accumulation of unfolded or misfolded protein in the ER lumen
triggers the increase in transcription of genes encoding ER
chaperones and other folding catalyses.

This splicing occur in the cytosol

HAC1 is transported back to the nucleus and

activates the transcription of ER-localized
chaperones to participate in binding of unfolded
proteins.

Only properly folded and assembled proteins get to leave ER.

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V. quality control:
Most mis-folded proteins are transported back out the ER (some through
the Sec61p translocon, some through a newly identified channel
called Derlin-1) and degraded by the ubiquitin proteosome system
in the cytosol.
Figure 6. Model for US11-mediated retro-
translocation of MHC class I heavy chains.
US11 recognizes HC in the ER lumen and
targets it to Derlin-1, a proposed component
of the retro-translocation channel. The p97
ATPase complex is recruited to Derlin-1 by
VIMP. HC emerging into the cytosol is
bound by p97. Poly-ubiquitin chains (Poly-
Ub, red) are attached and recognized by
both the N-domain (N) of p97 and the
cofactor Ufd1/Npl4 (U/ N). ATP hydrolysis
a virus by p97 moves HC into the cytosol. The
protein (to retro-translocation of misfolded ER proteins
destroy cell’s may occur similarly, with US11 being
antiviral replaced by other targeting components.
defenses) NATURE |VOL 429 | 24 JUNE 2004 p841-847

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 16.4 Export of bacterial proteins--all post-translational
Two major parts:
1. Across the inner membrane: to be inserted into the inner or outer
membrane or trapped in the periplasmic space.
2. Across both the outer and inner membranes and to be secreted the
extracellular space or injected into other cells
1. Across the inner membrane: similar to ER membrane translocation
(but post-translational)
So for all the systems we talk about today, we will ask 4
questions:
1. What is the nature of the targeting signal? Similar to
ER signal peptide (inter-changeable), cytosol
cleavage by signal peptidase (also
inter-changeable). Homologues for SRP periplasmic
and SR are also involved, but only used for space
post-translational insertion of very
hydrophobic membrane proteins into the S-S bond formation and folding
inner membrane.
2. What is the receptor? SecA (and SecB)
3. What is the structure of the channel? SecYEG, SecA uses the energy of ATP hydrolysis, which causes a
similar to Sec61!"# conformational change in SecA, to actively push the
Current Sec61 structure is actually from an archea. polypeptide through the SecYEG channel. A pulling
mechanism from the periplasmic space won’t work because
4. What is the source of energy? ATP, not GTP ATP will diffuse away through the outer membrane.

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2. Across both the outer and inner membranes and to be secreted into the extracellular space or injected into
other cells: divided into 4 types (I, II, III, IV)
Type III: Used by some pathogenic bacteria to inject toxins into host cells.

1. What is the nature of the targeting signal? An

amphipathic sequence at the N
terminus (for YopE)
2. What is the receptor? Small chaperone
proteins
3. What is the structure of the channel?A syringe-
like structure with similarity to
bacterial flagellum
4. What is the source of energy? ATP

Needle complex structure by cryoEM at 17 angstron from

Salmonella typhimurium
Fig. 1. The needle complex and the base complex of the TTSS from S.
typhimurium can adopt different symmetries in vivo. (A) Nomenclature
of the structural features of the needle complex.The needle complex is
divided into two distinctive substructures: the membrane-embedded
base and the extracellular needle filament. The base spans the
periplasm and is associated with the inner and outer membranes, where
ringlike structures are visible in electron micrographs of negatively
stained needle complexes (2% phosphotungstic acid, pH 7) (B). The
outer membrane– associated rings (OR1 and OR2) are composed of the
protein InvG, and the inner membrane– associated rings (IR1 and IR2)
contain the proteins PrgH and PrgK (4). The only protein identified
for the needle filament to date is PrgI (4). Bar, 30 nm. (C) Model-based
multireference alignment revealed significant differences in the
diameters of the average projections obtained for different
rotational symmetries, as indicated by white arrows in the comparison
of the IR1 of the 19- and 22-fold particles. (D) Distribution of different
symmetries in needle complexes isolated from wildtype S. typhimurium.
The data were generated by examining 3577 particles. (E) After sorting
of the particles and 3D reconstruction without enforcing any symmetry,
the true rotational symmetries could be derived from cross sections
through IR1 of the reconstructed needle complexes, as shown
for the 20- and 21-fold particles.
5 NOVEMBER 2004 VOL 306 SCIENCE p1040-1042 24
16.5 Protein sorting to mitochondria and chloroplasts--all post-translational.

Both organelles have N terminal cleavable signals. Comparing with the other two signals:

Mitochondrial targeting signal: amphipathic !-helix (positively charged amino acids on one side,
hydrophobic amino acids on the other side.

For “electrophoresis”+ o Hydrophobic

across the inner + o interaction with 25
membrane + o Tom20

Receptors/ channels:
Tom/Tim (translocon of the outer/inner membrane)
First identified by antibody inhibition, then by
genetic (confirm previous ones with knockout,
identify new ones by suppressor), now by mass
spec. or genomic approach.
General outline of the import process
e.g. for matrix proteins: Tom20/Tom22 receptor-->
Tom40 channel-->contact site-->Tim23/Tim17
channel-->Hsp70/Tim44 ratchet
Several important features:
1. Precursor translocated in unfolded form Tim50
2. Translocate through contact site
3. Energy: ATP on the outside for unfolding, in the +++
matrix for ratcheting precursors. PMF (400,000 _
V/cm) at the inner membrane for __
“electrophoresis” across the inner membrane

Translocate through contact site

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 Current model

Evolution of the Molecular Machines for Protein Import into Mitochondria

Pavel Dolezal, Vladimir Likic, Jan Tachezy, Trevor Lithgow SCIENCE (2006) VOL 313 p314

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Multiple signals and pathways targeting proteins to submitochondrial compartments

Tom70 28
Protein import into chloroplasts
Similarity to mitochondria
a Toc (translocon of outer membrane of chloroplasts)
complex with a receptor and a channel
a Tic complex with a connector and a channel
Energy: ATP in the intermembrane space and stroma,
possibly for Hsp70 but not sure yet. GTP is only
demonstrated by GTP-r-S inhibition.
Biochem. Cell Biol. 79: 629–635 (2001)
4 pathways to thylakoid, 3 have bacterial homologous
pathways
2 for thylakoid lumen:
$pH (Tat) for folded protein, use $pH
SecA for unfolded protein, use high ATP and
SecY
2 for thylakoid membrane
SRP: for LHCP, use GTP as energy and SecY
and Alb3(Oxa1) as membrane receptor
spontaneous
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 2 | MAY 2001 | 350-356

SRP/SR for post-

translational
insertion of very
hydrophobic
membrane proteins
into the inner
membrane.

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Traffic 2001; 2: 245-251

16.6 Protein import into peroxisomes: single membrane, no DNA, catalase as marker enzyme, most
abundant in liver (1-2 % of the liver cell volume).

1. What is the nature of the targeting signal? For most matrix proteins: PTS1: C-
terminal SKL, not cleaved after import. For a few other matrix
proteins: PTS2, approx. N terminal 9 amino acids, may or may not be
cleaved. Membrane proteins use different system (see below).
2. What is the receptor? Pex5 (PTS1), Pex7(PTS2) converge on the membrane
docking site Pex14/13/17, structure unclear. Pex5 partially or fully
enters the matrix, releases the cargo then recycle back to the cytosol.

3. What is the structure of the channel? Pex 10/12/2, structure not clear. Peroxisome can import folded proteins,
even 4–9 nm gold beads coated with albumin bearing PTS1s and microinjected into cultured cells. However,
no pore structure like the nuclear pore exists. Require transporter proteins for ions and proton passages--so
it’s totally sealed.
4. What is the source of energy? ATP, but don’t know for what.

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?
2 pathways to produce new peroxisome: de novo synthesis, and division (other
organelles like mitochondria and chloroplasts do not have de novo synthesis)

de novo
(Probably from division
ER membranes)

Peroxisome membrane and matrix proteins

are imported by different pathways

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