Lecture unit 2: Biosynthesis and

vectorial targeting of secretory

and
d membrane
b
proteins
i Part 1

Overview of the biosynthesis and sorting problem: Questions

to be addressed will include:
1. How were the sites of synthesis and subsequent
processing of secretory and membrane proteins experimentally
id tifi d?
identified?
2. What is the evidence that proteins are usually
inserted into the lumen of the ER co translationally?
3. What is the molecular basis for targeting mRNAs to
the ER versus free cytosolic polysomes?
4. What determines the precise trans bilayer
topography of integral membrane proteins?

Overview of the biosynthesis

y
and sortingg p
problem: Questions
to be addressed will include:
5. How does the ER translation machinery distinguish
between a secretory or resident luminal compartment protein
from an integral membrane protein?
6. What types of co and postranslation processing of
secretory and
d membrane
b
proteins
i occur, where
h
d these
do
h
occur,
and what are the functions for such processing events?
7. Once synthesized in the ER, how is a given protein
targeted to its proper destination?

Rough
R
h endoplasmic
d l
i reticulum
i l
mediated
di d biosynthesis
bi
h i off secretory,
plasma membrane integral proteins, resident organelle lumenal
and membrane proteins

1. In addition to plasma membrane integral proteins and

secretory proteins what organelle membrane proteins and
resident
id t luminal
l i l proteins
t i are synthesized
th i d on/in
/i RER
RER.
2. What organelle proteins are not synthesized/inserted at
the RER?

The secretory/ exocytic

(and endocytic) pathways

Traffic from compartment to

compartment is mediated by carrier
vesicles
l that
h bud
b d off
ff from
f
the
h donor
d
compartment and fuse with the
p
compartment.
p
These vesicles
recipient
carry both membrane and luminal
cargoes.

Diagram and rotary

shadowed image of
polyribosomes

A common pool of ribosomal subunits assemble both cytosolic

and ER bound polyribosomes.

Rough endoplasmic reticulum; thin section TEM.

TEM ER Lumen

En face view of polyribosomes on cytoplasmic surface

of the RER. Note spiral arrays of ribosomes of varied
length

What TEM method was used to generate this 3 D view of the ER?

Experimental dissection of the secretory pathway

Pulse chase analysis of the synthesis and sorting of
secretory
t
proteins
t i by
b the
th exocrine
i pancreatic
ti acinar
i
cell.
ll
Acinar cells synthesize and store in secretory granules a
large variety of digestive enzymes. After a meal, a peptide
hormone stimulates release (via exocytosis) of these
enzymes into ducts connected to the small intestine.

LM
histologically
stained section
of a pancreatic
acinus.
What are the
darkly stained
granules?
Pancreatic duct
for delivery of
digestive
enzymes to
small intestine

Why was the

pancreatic
acinar cell a
good system
to investigate
the secretory
pathway?

The arrow tracks the temporal and spacial route

taken by newly synthesized secretory proteins.

Thin section
TEM of the
RER, Golgi
region of a
pancreatic
acinar cell.

Cartoon depiction of a pulse chase experiment. There is continuous

flow (e.g. of newly synthesized protein) from compartment A >D. To
track temporal and spacial aspects of flow, a label (e.g. labeled amino
acid) is added to the system for a brief time (The pulse) and its fate in
space at various times after removing the label is tracked
tracked.

RER
Golgi
Condensing vacuole;
Secretory granule

Pulse chase analysis of secretory protein synthesis in the pancreatic

acinar cell
1. Cut pancreas into thin slices. Why is thin important?
2 Incubate slices in medium containing 3H (or 14C) leucine.
2.
leucine for ~ 3 min.
min
THE PULSE
3. Remove slices from pulse medium and place into new medium with
excess cold leucine
4. At various times remove slices and either a) process for thin section
EM or b) cell fractionation/organelle isolation. THE CHASE.

5a Cut thin sections,

5a.
sections cover with film emulsion and develop
after 2 4 weeks to visualize exposed silver grains (grids are
stained with uranyl acetate and lead after developing film)
or
5b. Place organelle fractions in scintillation counter and
quantify levels of radioactivity.
radioactivity
6. Quantify numbers of silver grains over RER, Golgi secrotory
granules as a function of time.

Diagram of an EM section preparation for AR analysis

0 chase
h
20 chase

45 chase
h
120 chase

Cartoon of pulse chase results. Silver grains are shown in red

pulse was 0 3 min

Condensing vacuoles

Initial steps in the co translational targeting and translocation of a

nascent polypeptide chain into ER lumen; from Pollard and Earnshaw.

Figure 13.6 Lodish et al. Cotranslational

translocation.

1. Translation begins in cytosol; signal sequence emerges from ribosome. 2.

Signal Recognition Particle (SRP) binds to signal sequence and arrests
p
4. SRP releases and SRP receptor
p
translation. 3. SRP binds to ER SRP receptor.
promotes insertion of N terminal signal sequence (as hairpin loop) into
translocon pore which is locked into open state by ribosome binding.

5. ER membrane bound signal peptidase cleaves off

N terminal signal
g sequence.
q
6. Cotranslational
insertion of the growing chain continues. 7 and 8.
Chain termination results in ribosome release from
ER discharge of completed chain into ER lumen and
ER,
closure of the translocon pore.

EExperimental
i
l characterization
h
i i off RER mediated
di d synthesis
h i and
d
insertion of secretory proteins:
Use of isolated rough microsomes isolated from secretory cell for
in vitro protein synthesis assays:
Demonstration that proteins are inserted into RER
lumen co not post translationally
Determination that the Signal sequence is generally
cleaved
l
d co translationally.
l i
ll
Identification and characterization of the signal
recognition particle.
Characterization of an aqueous pore in RER membrane
through which the nascent polypeptide is cotranslationally
inserted.
inserted

Tool kit needed for these studies

1. Isolated rough ER vesicles (rough microsomes)
2 Polyribosomes isolated from rough microsomes
2.
3. Stripped microsomes derived from rough
microscomes by removing polyribosomes from rough
microsome
i
membrane
b
surface.
f
4. In vitro protein synthesis system.

Preparation of
microsome and
cytosolic fractions
from tissue
homogenate by
differential
sedimentation.

microsome pellet is
composed of
vesiculated elements
of plasma membrane,
RER SER , endosome
RER,
and Golgi.

Rough and smooth microsomes are then separated by sucrose density

gradient sedimentation.

Thin section TEM of RER and a pellet of RER derived rough

microsomes.

RER

rough microsomes

Thin section
TEM of rough
microsomes
and
d

polysomes
detached from
ro gh
rough
microsomes
using non ionic
detergent.

Experimental dissection of
membrane
b
and
d secretory
t
protein biosynthesis:
evidence for rapid insertion
into RER lumen:
ee.g.
g pulse label tissue slice,
slice
then immediately isolate
microsomes; add protease,
no digestion
di ti off newly
l
synthesized protein.

If control doesnt work, need to select

other protease

Isolation of strippedpolysome
stripped polysome free microsomes from rough
microsomes:
1. Extract rough microsomes with high salt and puromycin.
What is the purpose of the puromycin?
2. Collect stripped microsomes by high speed sedimentation.

Evidence for co translational insertion:

Experiment 1:
a)

Isolate rough microsomes from cells/tissue making predominantly single

protein

b)) isolate stripped

pp microsomes ((usingg salt and p
puramycin)
y ) and c)) detached
polysomes (by detergent treatment)
c)

Perform in vitro translation assays using 35S methionine to identify

synthesized products.
products The following assays are performed:

Stripped
microsomes

Rough
R
h
microsomes

Detached
polysomes

Assay 1: Rough microsomes (RM) + protein synthesis cocktail (PSC)

Assay 2: Detached polysomes (DP) + PSC
Assay 3: DP+ SM+ PSC
After 30 minutes split each asay mix into 2 aliquots; treat one with
protease. Analyze the 6 aliquots from the 3 assays by SDS PAGE
autoradiography (AR): Result:

RM

DP
Note
higher
Mr

Processed
and
protected

SM+DP

Processed
and
protected
+

+ protease

Text

Experiment 2: Does protease protection (luminal insertion)occur co or

post translationally?
Assay 1: DP
DP+SM+
SM PSC (same as above; control)
Assay 2: a) DP + PSC for 30 minutes;
b) add protein synthesis inhibitor
c) add SM.
d) split into two aliquots and treat one with protease: SDS PAGE AR
Conclusion: completed proteins cannot insert post translationally
Assay 1

Assay 2
Note
higher
Mr

+ protease

Evidence that removal of the signal sequence is cotranslational

1. Isolate stripped polysomes (as before from cells making mostly a single
secretory
t
protein)
t i )
2. Use as substrate for in vitro protein synthesis under conditions that
only nascent chains can be completed, but new chain initiation is
prevented.
d
3. Remove aliquots at various times and analyze the finished products by
SDS PAGE AR:

5
5

Green portion of
completed chain is
radioactive

3
3

Step 1

3 2

Nascent chains

Step 2
1 2

completed chains

State of polysomes isolated from rough microsomes

if processing were Co or Post translation.
5

Cotranslational
removal of signal
sequence

Post translational
removal of signal
sequence

Evidence for co translational removal of

N terminal signal
g
sequence.

Last nascent chains

h
completed were not
processed. i.e. were
p
not long enough for
signal peptidase to
cleave off signal
sequence.

First nascent
chains completed
are already
processed; i.e.
signal sequence
was removed
d prior
i
to isolation of
rough microsomes
1

4 10 15 20 30 min.

Evidence for co translational processing of the N terminal

signal sequence from secretory proteins: Blobel and
D bb t i JCB:
Dobberstein:
JCB 1975
This AR shows the synthetic
products from polysomes
added to in vitro protein
synthesis assay under
conditions that only allows
completion of attached
nascent chains. Note that
earliest time points the signal
sequence has been removed.
The pre form of Ig light chain
appears only in later time
points.

Diagram what this gel

would look like if
removal of signal
sequence were post
translational.

Examples of some signal peptide/sequences:

+ basic aa
~10 30
hydrophobic aa

How would yyou experimentally

p
y demonstrate
that the signal sequence is both necessary and
sufficient for targeting secretory proteins to the
lumen of the RER?

1. Translation begins in cytosol; signal sequence emerges from ribosome. 2.

Signal Recognition Particle (SRP) binds to signal sequence and arrests
p
4. SRP releases and SRP receptor
p
translation. 3. SRP binds to ER SRP receptor.
promotes insertion of N terminal signal sequence (as hairpin loop) into
translocon pore which is locked into open state by ribosome binding.
Co translational=happening while protein is made

SRP has several distinct functions mediated by different SRP

protein subunits: a) signal sequence binding; b) translation
arrest c) SRP receptor binding and d) GTP binding/hydrolysis

Experimental characterization of SRP translation arrest

properties:
Assay synthesis (using in vitro protein synthesis cocktail) of
detached polysomes (or reconstituted polysomes using mRNA
encoding secretory protein of choice; e.g. pre prolactin ) in the
absence and presence of SRP:
+ SRP

The 70 aa peptide
Th
tid
represents the ~ 40
aa within the
ribosome pore and
~30 aa of SS
sequence protruding
from large ribosomal
subunit:

Pre prolactin
~ 70 aa peptide

Evidence for an aqueous channel for chain translocation: release of

nascent chain from ribosome with p
puromycin
y creates a ion p
permeant
channel that closes upon release of ribosome from membrane.

puromycin
p
y mimicks an
incoming aa tRNA but
cannot form a peptide
bond and thus causes
release of the nascent
chain from ribosome,
opening aqueous
channel in ER
membrane

A lipid bilayer (called a black lipid film) is formed across a small hole in
a plate and then RMs are added to one side of the plate; the only
route:
route: for current/ion flow is through the lipid film.
film

How would you

demonstrate that the
effect of puromycin is
specific; e.g. maybe it is
altering bilayer
permeability.

The ribosome is critical for maintaining the open state.

Salt removal of ribosomes results in channel closure.

Models
M
d l for
f co ttranslational
l ti
l iinsertion
ti off
secretory and membrane proteins through
th RER membrane
the
b

Insertion and processing of a secretory protein

Insertion of Type 1 single spanning transmembrane protein

with a cleavable signal sequence.

Insertion of Type 2 single spanning transmembrane protein

Internal start transfer and stop transfer sequences for insertion of

multi membrane spanners.

IInternal
t
l signal
i l sequence insertion
i
ti off Type
T
1 single
i l
spanning transmembrane protein; no signal sequence
cleavage.

Start and stop transfer sequences in a multi spanning

membrane p
protein; these sequences
q
become the spanning
p
g
domains in the completed protein

Only start
requires SRP

Determination of integral membrane protein topography during

biosynthesis on the RER:
Key determinants:
1. N terminal signal sequence consisting of a hydrophobic core of
amino
i acids
id Generally
G
ll contains
t i consensus sequence for
f cleavage
l
b
by
signal peptidase C terminal to the hydrophobic core. This forms a
loop which promotes transfer of growing chain C terminal to the
signal sequence. diagrammed here as N SSSSS
2. Internal hydrophobic stop
2
stop transfer
transfer sequence segment
segment
consists of a hydrophobic , alpha helical segment that arrests chain
transfer and becomes a transmembrane segment in the completed
protein.
t i These
Th
are preceded
d d by
b either
ith an N terminal
t
i l signal
i l
sequence or an internal start transfer sequence. diagrammed here
as XXXXX

Determination of integral membrane protein topography during

biosynthesis on the RER:
3. Internal start transfer sequence: Like the N terminal signal
sequence, consists of a hydrophobic segment flanked on either the
N or C terminal side by charged (generally +) amino acids. This will
promote chain insertion of protein chain segments either C terminal
(+ charge on N terminal side of hydrophobic core) or N terminal (+
charge on C terminal side of hydrophobic core) to the start transfer
sequence. +TTTTT or TTTTT+

Sequence key for protein insertion demo

Protein 1:
Protein 2:
Protein 3:
Protein 4:
Protein 5:
Protein 6:

N-SSSSS-----------------------------------------C
N-SSSSS-----------------XXXXX--------------------C
---------------------TTTTT+------------------------C
----------------------+TTTTT----------------------C
----------------+TTTTT-----------XXXXX--------C
N-SSSSS---------XXXXX---------------+TTTTT----------C