CRITICAL ANALYSIS

PRESENTED BY
AROMAL NATH V S
2nd semester
Msc Biotechnology
CUSAT
 2013

WILLIAM E. BALCH WILLIAM E. DUNPHY WILLIAM A. BRAELL JAMES E. ROTHMAN

The Nobel Prize in Physiology or Medicine
2013
Backstage knowledge…
• The classic work of George Palade had made it evident that
the secreted proteins are carried from the endoplasmic
reticulum (ER) to the cell surface in specialized containers, or
transport vesicles, that bud from one membrane and fuse with
the next, transiting the Golgi stack on the way.
CONTD.
• In early 1960s “coated vesicles”
were discovered by Thomas F.
Roth and Keith Porter while they
were analyzing electron
micrographs of mosquito
oocytes.
• The first protein (clathrin)
coating vesicles budding from
plasma membranes were
purified by Barbara Pearse in
1976.
CONTD.
• Gunter Blobel discovered in
1975 that proteins carry built-in
signals that direct them into the
ER and by the implication that
proteins quite generally have
signal sequences that specify
their location in the cell, for
which he got Nobel Prize in
1999.
CONTD.
• Michael Brown and Joseph
Goldstein was able to elucidate
the basic mechanisms of
cholesterol homeostasis in
1970s.
• They found that clathrin-coated
vesicles garner lipoproteins from
the medium by means of a LDL
receptor localized to the coated
regions of membrane involving
in budding.
 GAP GAP
As a result of all of this,
it became evident that
solving the SORTING
PROBLEM essentially GAP
GAP required
UNDERSTANDING how
each type of vesicle
targets to and fuses
with the correct target
membrane in the cell
GAP
GAP
Hypothesizing the problem in a unique
way…
• He set the mindset of a “The anatomical arrangement of
endomembrane system in cell is
physicist! vital to ensure the delivery of
• Other cell biologists: cargo proteins”

• James Rothman:

“My idea is simple, but opposite: the

INTRINSIC CHEMICAL SPECIFICITY enables
specific cargo delivery and the observed
anatomy arises as the consequence of
chemical specificity in operation or simply
SPECIFICTY DICTATES ANATOMY”
CONTD.
 Thecell-free
The model system…
reconstitution of the vesicle
transport
• The thought had begun since
1970s, and Rothman survived all
type of skeptic attitude of the
scientific community against the
reconstitution experiment.
• He has published a series of
research papers in which all the
time he has improved at least
some technical methods.
CONTD.
Sorting Packing
• He looked for the transport of
protein between the successive
Tagging
compartment of the Golgi.
• The Golgi is well suited for a
functional reconstitution
because its stack can be isolated
intact.
• The specific glycoprotein (G
protein) transport was of their
interest.
 CONTD.
• The G protein was abundantly expressed
during infection by Vesicular Stomatitis Virus
(VSV).
• The Asn-linked oligosaccharide addition on to
G protein was done by Glucosidase I in the
ER.
• These N-linked G protein was modified with
the addition of GlcNAc by N-
acetylglucosamine transferase I enzyme in
the medial-Golgi.
• This modified G protein is resistant to
cleavage by Endoglycosidase H (Endo H), a
microbial enzyme.
VSV-infected mutant CHO clone 15B cells
&
VSV-uninfected wild-type CHO cells
CONTD.

ATP (or other NTP) is required for the transport of protein.

Pre-existing cytoplasmic organization is not important for accurate transport
between Golgi compartments.
Methods employed…
Preparation of cells, Virus and Antiserum
• The wild-type line of CHO cells was
maintained in suspension and the
CHO mutant, clone 15B cells was
grown in monolayers.
• Stock of VSV (vesicular stomatitis
virus) was grown in monolayers of
BHK cells.
• Purified virions were used as
antigen in rabbits for preparation of
anti-G protein antiserum.
• Preimmune serum was also
collected.
 Infection of 15B cells to yield Donor
fractions
1) Densely confluent clone 15B
cells were infected with VSV in
serum-free growth medium
containing an antibiotic & buffer.
1 hr
Infection
2) A complete growth medium was
added.
At 3.5 hr

3) The cells were removed by

trypsinization
Homogenization of cells
• For donor & acceptor fractions a
crude homogenate was prepared
in sucrose solution with both
Dounce homogenizer and Ball
homogenizer, then suspended in
homogenate buffer.
• Ball homogenizer
SuspensionSyringesPrecision
boreStainless steel ballCell
breaks.
• Crude homogenate was frozen in
liquid nitrogen at -80°C.
 Preparation of donor & acceptor Golgi membrane fractions by dens ity gradient centrifugation

1) A postnuclear supernatant (PNS) was

prepared by centrifugation at 600*g for 5
min (for PNS assay)
2) Crude homogenate + ice-cold sucrose +
Na2EDTA
3) Vortexed vigorously
4) Overlaid with sucrose-HCl
5) Centrifuged at 25000 rpm for 2.5 hrs
6) The turbid band at the sucrose Interface
III was harvested by syringe puncture
(donor or acceptor fractions are obtained
usually at these concentrations).
7) Protein was measured by the Lowry
method.
 Preparation of high speed supernatant
fractions (cytosol)
1) Homogenate from uninfected
wildtype 15B CHO cells centrifuged
at 49000 rpm for 60 min
2) Supernatant filtered by gel
filtration method* buffered with
Tris-HCl
3) The void volume fractions,
containing all the excluded protein,
were collected, pooled and filtered.
*This is to remove the inhibitory low
molecular cytosolic pool of UDP-GlcNAc
CONTD.
Acceptor
membrane

Donor Cytosol
membrane fraction

Incubation conditions
Incubation conditions to achieve transport In
Vitro
BUFFER SALT STOCK SOLUTION REGENERATING SYSTEM
• HEPES-KOH • CPK
• KCl • CP
• MgOAc • ATP
• Na form, neutralized with NaOH
• UTP
• H2O with UDP-3H-GlcNAc
CONTD.
Acceptor
membrane

Donor Cytosol
membrane fraction

Incubation at 37°C for 60 min

Incubation conditions
 Quantitation of transport by the
incorporation of 3H-GlcNAc into G protein

Detergent Buffer
Tris-HCl
NaCl
Na2EDTA Membranes
Incubation
Triton X-100 solubilized
1% Na Cholate
 Scintillation cocktail

CONTD. Preimmune
serum

Anti-G
Antiserum

Solubilized
45 min The amount of [3H]-G
membranes protein produced was
37°C immunoprecipitated on
to Millipore filters
Result Analysis…
Transport-coupled incorporation of 3H-
GlcNAc into G protein
• Increasing time
• Preimmune serum : Only trace retention of
3
H-GlcNAc
• Anti-G serum : Increment in the retention of
3
H-GlcNAc
• Immunoprecipitate was formed after 45 min
SDS gel electrophoresis
• Lane a : G protein is the major protein
labelled among all protein species
• Lane b: G protein as the only radiolabeled
protein species in immunoprecipitate
• Lane c : No radiolabeled protein species with
preimmune serum
Density gradient centrifugation
• Sucrose gradient with Tris-HCl
• Density increases from top to
bottom
• 3 interphase bands of concern
• Pellet formation and washing
steps have avoided, as
membranes of concern was got
on the interphases
Copurification of donor activity with Golgi
markers
• Donor activity (ability to provide G
protein) of Golgi membranes from
VSV infected 15B cells
• Proteins which are unbound
• Each gradient fraction assayed by
copurification for the activity of:
1) Glucosidase I :ER marker
2) Mannosidase I : cis Golgi marker
3) Galactosyltransferase : Golgi
marker
• Thus donor activity was purified 20
fold over PNS
PNS fraction v/s Donor Golgi fraction

Specific activity
Copurification of acceptor activity with Golgi
markers • Acceptor activity ( H-GlcNAc incorporated
3

at 60 min) of Golgi membranes from

uninfected wild type cells
• Proteins which are unbound
• Each gradient fraction assayed by
copurification for the activity of:
1) Glucosidase I :ER marker
2) Mannosidase I : cis Golgi marker
3) Galactosyltransferase : Golgi marker
4) GlcNAc transferase I : medial Golgi
marker
• The acceptor activity was purified about
9 fold over PNS
PNS fraction v/s Acceptor Golgi fraction

Specific activity
Major conclusions of the work…
• Cell-free system is highly specific.
• It can measure only transport between the sequential
compartments in the Golgi stack.
• They were able to quantify the transported protein.
• Transport in vitro is almost as efficient as in the cell.
• It requires ATP and the cytosol fraction in addition to protein
components on the cytoplasmic surface of the Golgi membranes.
• The copurification strategies have made the assay more evident
for the specificity of the transport.
Interesting aspects…
• This reconstitution assay makes a remarkable prediction:

“Accurate vesicle traffic can take place

in cell-free extracts, which would open
the door to Kornberg-style enzymology.
Once reconstituted, cell-free transport
could be used as an assay to permit the
underlying enzyme proteins to be
discovered and purified according to
their functional requirements”
CONTD.
 Autoradiography of
electron microscope
The great paper… sections

Electron microscope
immunocytochemistry
Stepping stone
of discoveries in
vesicle
trafficking GTPase switch mechaism

NSF, SNAP, SNAREs- Fusion machinery

THANK YOU