membrane topology (review)

protein trafficking overview

ER intro

1
Membrane topology (review), protein trafficking overview, ER intro:

The faces of
cellular
membranes:
the exoplasmic
and cytosolic
leaflets of
membranes
differ in
composition
and function
Related Know: Cytosol vs. Cytoplasm
AND logic of referring to lumen of compartments in
170828_Maner-Smith_Biomembrane the secretory pathway as topologically extracellular2
Structure.pptx sl11
Membrane topology (review), protein trafficking overview, ER intro:

Major protein-sorting pathways in eukaryotes

3
Membrane topology (review), protein trafficking overview, ER intro:

Our focus today: the ER; moving proteins into the ER

4
Are secreted proteins translocated to the ER / are they proteolytically processed when translocated?

Structure of the rough ER

Mol. Cell Biol. 7th ed. Fig. 13-2 5

 Preparing an endoplasmic reticulum
fraction:"microsomal fraction"

6
Preparing an endoplasmic reticulum fraction:"microsomal fraction"

Methods: Breaking cells and tissues

Key terms here (in magenta): homogenization, homogenate, extract

Note assertion: When carefully conducted, homogenization leaves most of the membrane-
enclosed organelles largely intact 7
Preparing an endoplasmic reticulum fraction:"microsomal fraction"

Cell fractionation by differential centrifugation

8
Preparing an endoplasmic reticulum fraction:"microsomal fraction"

Isolation of purified rough and smooth microsomes

by centrifugation of a cell homogenate through a
sucrose gradient EM appearance of the
rough ER fraction
(rough microsomes)

The circular structures are

microsomes

9
 Phospholipid synthesis occurs on the ER
membrane
(important topic and now good time to introduce this but admittedly a
side excursion not directly related to todays main story )

Why talk about this now? Some reasons:

Much of the focus of Block 4 is related to the membrane bound compartments of cells
and the plasma membrane.
This discussion raises questions regarding one aspect of how these compartments are
made
Happens that our current understanding is that most of the lipids of all the membrane
bound compartments is made in the endoplasmic reticulum (ER)
One experiment we will discuss in this section introduces an assay to be used
repeatedly later in the discussion (microsomes treated with protease; the
interpretation of such protease protection studies). 10
Does phospholipid synthesis in ER / phospholipid translocation

Most phospholipid synthesis occurs in the ER

11
 Does phospholipid synthesis in ER / phospholipid translocation

Do these data support the hypothesis that

the biosynthesis of phosphatidic acid,
phosphatidylethanolamine, and
phosphatidylcholine occurs on the cytosolic
side of the ER?
1 2 3
Control. Known to be
luminal 4 4

5,6

2
Ignore this 3

4
5
Ignore this

Deoxycholate and taurocholate are detergents

Data from Coleman, R. and Bell R.M. (1978) J. Cell Biol. 76, 245 12
Does phospholipid synthesis in ER / phospholipid translocation

What is mechanism by which lipids move between compartments and

membrane leaflets?

No exchange protein

Exchange from liposomes (no flippase)

Data from Zilversmit1977_Biochim Biophys Acta469_99 +

Exchange from microsomes (flippase)

some hypothetical data (blue, red line)

Rat liver microsomes were labeled with 32P
Microsomes were incubated with excess mitochondria
The amount of labeled phosphatidylcholine in the microsomes was determined as a function of
time

Others have shown that phospholipids added to the cytoplasmic side of microsomes are rapidly
flipped to the lumenal surface.
This process is saturable and inhibited by protein modification reagents suggesting that it is
protein mediated. 13
Protein (flippase) in the ER has not been identified.
Does phospholipid synthesis in ER / phospholipid translocation

Schematic of Zilversmit and Hughes Experiment

Start Finish
No exchange
protein

Labeled Excess
microsomes mitochondria

With
phospholipid
exchange With flippase
protein

Labeled Excess
microsomes mitochondria

No flippase

14
 For the remainder of
this class we will draw
heavily on the work of
Gunter Blobel

Blobel prize: https://www.nobelprize.org/nobel_prizes/medicine/laureates/1999/

Blobel lab: http://www.rockefeller.edu/research/faculty/abstract.php?id=225#content 15
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Do proteins that are secreted enter the ER?

Do secreted proteins get proteolytically processed
co-translationally?
Are secreted proteins proteolytically processed
when translocated?
If there is a cleaved signal peptide is it at the N-
terminal end of proteins trafficked into the ER?
Does translocation need to be co-translational?
Can translocation activity be reconstituted in
proteoliposomes?
Does translocation occur through a channel? 16
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Do proteins that are secreted enter the ER?

Rough

+ differential (and sucrose-density gradient centrifugation?)

17
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Cells and tools for experiments of next slides

Myeloma cell line used
myelomas are tumor cells that derive from plasma cells. Plasma cells differentiate
from B cell lymphocytes and are specialized to produce large amounts of
immunoglobulins.
the myeloma cell line used produces (and secretes immunoglobulin light chain but
not heavy chain

Rough ER (rough microsome) fraction prepared from the myeloma cells

(homogenization, differential and/or gradient centrifugation)

Detached polysomes
Rough ER treated with gentle detergent to dissolve the microsomal membrane
Polysomes = mRNA with the multiple ribosomes attached. (polysome images on
next slide)

In vitro translation mix

Contains AAs, tRNAs, energy source (everything needed except ribosomes and
mRNA). For autoradiography, one or more of the AAs is radioactive.

In vitro translation protocols

Readout = let ribosomes on RNA (polysomes) finish translation
Initiation = start with naked mRNA + ribosomes subunits --> allow translation to
initiate. 18
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Polysomes: some images

Free polyribosome
in cytosol

ER membrane

Polyribosome
bound to ER
membrane by
multiple nascent
polypeptide chains We will look at experiments exploring
the ideas depicted here 19
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Do secreted proteins get proteolytically processed co-

translationally?
Lane 1: cell free translation of Ig
light chain mRNA (no msomes).

Lane 2: secreted Ig light chain (from

myeloma cell culture medium)

Lane 3: readout of detached

polysomes. Cells are labeled;
msomes solubilized; translation
initiation inhibited; translation
continued.

Conclusion: ??

Note: difference in apparent MW of Pre-light and

mature is approx. 3kD [ = how many AAs?]

20
Blobel, G. (2000) Protein Targeting (Nobel Lecture) Chembiochem 2000, 1, 86
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

If there is a cleaved signal peptide is it at the N-

terminal end of proteins trafficked into the ER?
Lane 1: cell free translation of
Ig light chain mRNA (no
msomes).

Lane 2: secreted Ig light chain

Lane 3-6: readout of detached

polysomes for increasing
amounts of time.
Cells are labeled; msomes
solubilized; translation initiation
inhibited; translation continued.

Conclusion: ??

Note: difference in apparent MW of Pre-

light and mature is approx. 3kD = how
many AAs

21
Blobel, G. (2000) Protein Targeting (Nobel Lecture) Chembiochem 2000, 1, 86
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Blobel and Dobbersteins

1975 sketches of their model

22
Blobel, G. (2000) Protein Targeting (Nobel Lecture) Chembiochem 2000, 1, 86
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Does translocation need to be co-translational?

How would you test if:
there is incorporation into
microsomes?
there is removal of the signal
sequence?

Conclusions: ??

23
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Can translocation activity be reconstituted in

proteoliposomes?
mRNA used encodes
Microsomes Proteoliposomes
preprolactin
vesicles = either microsomes or
proteoliposomes
proteoliposomes prepared by
solubilizing microsomes, then
reconstituting the proteins into
phospholipid vesicles.
PROT. K = proteinase K
TX-100 = triton X-100, a non-
ionic detergent

Conclusions: ??

Why bother?
(What can you use this artificial
reconstitution system for?)

24
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Bilayer technique
Does translocation occur through
a channel?

Predictions:
If translocation is through a channel, the
Scheme of puromycin experiment electrical conductance of the membrane will be
higher when channel is open.
If translocation just involves nascent peptide
sliding through membrane, no increased
conductance.

Trick: puromycin (which mimics aminoacyl t-RNA

can be added onto growing chain, but next AA cant
be added, so chain terminates, but in a fashion
such that the ribosome doesnt detach.

Addition of puromycin to the cis side

of the bilayer dramatically increases
current flow

Conclusion: the protein channel

does not conduct current while the
nascent peptide is in the channel

25
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Puromycin has no effect when added to

trans side.

By decreasing the puromycin concentration

single channels of 220 pS were detected

Addition of high salt removes

ribosomes and closes channels.

26
The Blobel lab experiments: Do proteins that are secreted enter the ER? (and onward)

Summary: Model of co-translational translocation in ER

Signal recognition particle (SRP) composed of 6 polypeptides + 7S RNA

Signal recognition sequence
Translational control
Targeting to ER (SRP receptor)
SRP Receptor is a heterodimer (SR alpha and beta)
Binding and release of SRP and its receptor is regulated by GTP cycle 27
MCB8 13-6 (p589) / MCB7 13-6 (p583)
End of part 1

28
Topology and Insertion of
Membrane Proteins

29
Topology and Insertion of Membrane Proteins
Case study: Aquaporin

Mechanisms by
which this
topology
achieved?
How determine
topology?

30
 Aquaporin: topological maturation?

ER How
investigate?

Plasma Shaded ovals:

hydrophobic segments
membrane that are putative TM
segments
Open circles:
Glycosylation at N42
Arrows:
Demarcate AAs to which a
reporter was added for the
experiments reported.
Lu et al. Mol Biol Cell 11:2973 31
Your problem: What can you infer
regarding the topology?
HR: hydrophobic region
Open circle: known glycosylation
site
Consensus for N-glycosylation is
N-X-S/T

Faint band at upper arrowhead in

lane 4 is real (and is important to the
interpretation)

Tripeptide inhibits N-linked

glycosylation.

32
 Positioning of type 1 single pass proteins

Mol. Cell Biol. 7th ed. Fig. 13-11

MCB8 13-11 (p595) / MCB7 13-11 (p588)

33
 Positioning of type II and type III single pass proteins
Type II
In prokaryotes Type II orientation is believed to be mediated
(most commonly by the positively charged residues shown
N-terminal to the signal-anchor sequence.
In eukaryotes positively charged residues N-terminal of the
signal-anchor sequence may be important in determining
Type II topology for some proteins but other features (still
being sorted out) are also important.
MCB8 13-12 (p596) / MCB7 13-12 (p589)
Type III
In prokaryotes Type III orientation is believed to be
mediated (most commonly) by the positively charged
residues shown C-terminal to the signal-anchor
sequence.
As for Type II, in eukaryotes other features important in
determining this topology for many proteins.
34
 Topogenic sequences determine the orientation of ER
membrane proteins

Can you provide a rationale for:

why some of these hydrophobic
stretches are called STAs and
some SAs?
for the ordering of SAs and STAs
in Type IV proteins
Suggest you draw some sketches to
see this.

MCB8 13-14 (p598) / MCB7 13-14 (p591)

35
 Hydropathy Profiles

MCB8 13-16 (p600) / MCB7 13-16 (p593)

36
Aquaporin: predicted topology
based on hydrophobicity

Note that Y-axis here is

hydrophilicity

37
Post-translational processing in the ER

Chaperones (BiP, Calnexin, Calreticulin) dependent

folding
Formation of disulfide linkages (PDI- protein disulfide
isomerase)
Glycosylation (form O-linked or N-linked oligosaccharides)
Attachment to GPI anchor

38
 N-linked
glycosylation
Common precursor
of N-linked
oligosaccharides

N-glycosylation
consensus
sequence

For 555 purposes, you dont need to know the details.

555 Take-homes (this and next two slides):
N-linked glycosylation is initiated in the ER.
The consensus sequence for N-linked glycosylation is N-X-S/T
At each N-linked glycan site in a protein, glycosylation begins with the addition of a
common precursor
Synthesis of the precursor involves the cystosolic and luminal leaflet with flipping of
components formed on the cytosolic face
After addition of the common precursor, modification may occur in the ER and Golgi

Compare/contrast: 39
In addition to N-glycosylation, what other kinds of protein glycosylation occur?
 Biosynthesis of N-linked oligosaccharide precursor

For 555 purposes, you dont need to know the details.

555 Take-homes:

40
Addition and initial processing of N-linked oligosaccharides

41
Mechanism of attachment
of GPI-anchored proteins

GPI: glycosylphosphoinositol

42
Now please go to work on your problem:
What can you infer regarding the topology of this
aquaporin construct?

HR: hydrophobic region

Open circle: known glycosylation
site
Consensus for N-glycosylation is
N-X-S/T

Faint band at upper arrowhead in

lane 4 is real (and is important to the
interpretation)

43
The End

44
 Appendix:
Supplemental information

45
 Reconstitution of translocation: early demonstration

For this experiment:

Translation mixture: As in
preceding experiments
mRNA: light chain Ig +
hemoglobin

Microsomes:
where present stripped
using EDTA (no attached
ribosomes initially)
source of microsomes: dog
pancreas

Translation protocol: initiation

Conclusions: ??

46
Blobel, G. (2000) Protein Targeting (Nobel Lecture) Chembiochem 2000, 1, 86