RECONSTITUTION OF THE TRANSPORT OF

PROTEIN BETWEEN SUCCESSIVE

COMPARTMENTS OF THE GOLGI MEASURED BY
THE COUPLED INCORPORATION OF
N-ACETYLGLUCOSAMINE

William. E. Balch
William. G. Dunphy
William. A. Braell
James. E. Rothman
NOBEL PRIZE (PHYSIOLOGY-2013) WINNING TRIO
(VESSICULAR TRANSPORT)
PROTEIN SORTING AND TRAFFICKING
• Sceretory, lysosomal and plasma membrane
proteins- secreted by ribosomes on ER.
• Elucidated by electron micrographs and pulse
chase.
• Signal peptides (10-15 aa – NH2 end).
• SRP docks ribosomes on ER.
• Protein trafficked through Golgi in vessicles.
PROTEIN TRAFFICKING THROUGH GOLGI
Golgi:
• Flattened, disk-like, membranous cisternae with dilated
rims and is a ribbon-like complex in appearance and is
adjacent to the nucleus.
• Diameter - 0.5 to 1 micron.
• Stacked on top of one another (typically fewer than 8
cisternae).
• 3 functionally distinct compartments- 1) Cis, 2) Medial and
3) Trans.
CONTD.
• Cis – Protein sorting, fatty acids addition, glycoside
trimming.
• Medial - PTM (GlcNAc addition).
• Trans – Galactose addition.
CONTD.
MODELS OF TRAFFICKING

1. Cisternal progression.
2. Retrograde vessicular transport.
INTRODUCTION
• Cell free systems need to be developed to study extensively, the
trafficking of vessicles.
• Golgi is well suited for reconstitution as the stacks can be
isolated and purified intact.
• There are also clear markers for Golgi.
• Golgi involved in protein transport, hence the components
needed for transport should be concentrated in the Golgi
fractions.
• Action of different glycosyl transferases makes is it easier to
study the transport between successive compartments.
INTRODUCTION
• Transport between Golgi compartments is a Vectorial and
Dissociative process.
• Transport vessicles can bud off one golgi population and
fuse to another golgi population.
• It’s a random process.
• Understanding: Transport vessicles sorted on the basis of
biochemical properties and not physical proximity.
CONTD.
• The old (previous) assay.
• VSV infected CHO 15B and uninfected CHO W.T cells are
used.
• G protein is tracked for its transport coupled GlcNAc
addition.
• Why was G Protein tracked?
The cell free system also respects the compartmental as in the in vivo system,
exhibiting appropriate specificity.
CONTD.

• In this paper, a rapid, more quantitative, flexible and a

sensitive form of assay, as well as an extensive purification
of active and stable donor- and acceptor- containing Golgi
fractions are reported.
NEW ASSAY

• Tritiated GlcNAc rather than S35 Met is used in the pulse

chase.
• Direct method fo assaying, whereas the old assay was an
indirect measure (Endo H resistance assay).
TRANSPORT COUPLED INCORPORATION OF
N-ACETYLGLUCOSAMINE
• OBJECTIVES:

 To develop a more RAPID, SENSITIVE and FLEXIBLE

assay.
CONTD.
• Crude homogenate of VSV infected 15B and wild type were made.
• Homogenate centrifuged at 600g for 5 mins.
1
• PNS from the two cells were co-incubated in the presence of ATP and
an ATP regenerating system.
2 • Assay terminated by detergent addition.

• Anti-G serum and Pre-immune serum were used to immuno-

precipitate the sample.
3 • The protein was counted for radioactivity with scintillation counter.
CONTD.
DONOR ACTIVITY COPURIFIES WITH THE GOLGI
MEMBRANES OF VSV INFECTED 15B
• OBJECTIVES:

 The improved assay should measure the donor activity

quantitatively.
 To prove donor activity indeed lies with the Golgi
bodies.
CONTD.
• Crude homogenate was prepared (VSV infected 15B Cells).
• Layered on a discontinuous density gradient centrifugation.
1
• Donor activity was tested for various fractions:
• PNS and Golgi fractions were co incubated with acceptor (Golgi from uninfected wild
2 type cells).

• The sub-compartmental markers (mannosidases, galactosyl transferases, etc.) were

also tested for various fractions.
3 • Recover, specific activity and fold enhancements were calculated for various fractions.
ACCEPTOR ACTIVITY COPURIFIES WITH THE
GOLGI MEMBRANES OF WILD-TYPE CELLS
• OBJECTIVES:

 The improved assay should measure the acceptor

activity quantitatively.
 To prove acceptor activity indeed lies with the Golgi
bodies.
CONTD.

• The assay was done as in the assay for checking donor

activity, with the modification that, Homogenate of
uninfected CHO Wild Type were layered on the density
gradient.
• The various fractions were assayed for acceptor activity
and also tested for the GlcNAc transferase activity.
GLYCOLSYLATED G PROTEIN IS A
TRANSMEMBRANE PROTEIN SEQUESTERED IN
SEALED GOLGI MEMBRANE VESSICLES
• Objective:

 To test whether the transferred G protein is present in a

sealed membrane compartment.
CONTD.
• Golgi fraction from CHO 15B cells were co-incubated with the same from wild type cells.
• The assay was terminated by the addition of detergent and run on SDS PAGE.
Lane 1
• Golgi fraction from CHO 15B was co-incubated with that from wild type cells and trypsinised.
• The reaction was terminated as above and was run on SDS PAGE.
Lane 2
• Golgi sample from both the cell types were incubated and trypsinised in the presence of triton
x 100.
Lane 3 • The reaction was terminated and it was run on SDS PAGE.
 They concluded from lane 2 that 95% of
radioactive GlcNac incorporated into the
oligosaccharides of G protein remained
attached to immunoprecipitable polypeptide
after trypsin treatment

 They also concluded from lane 3 that only

2% of the incorporated GlcNac
immunoprecipitated when treated with trypsin in
presence of Triton X-100

 Above results establish that the transported

G protein is retained in sealed golgi vesicles
with its carboxy terminal domain on the outside
and its oligosaccharide chains on the inside
RAPID DEPLETION OF G PROTEIN IN DONOR
COMPARTMENT WHEN PROTEIN SYNTHESIS IS
INHIBITED IN VIVO
• Objective:

 To confirm that the protein G is localised in the Golgi

and to study its depletion as a function of time when
protein synthesis is inhibited.
CONTD.
• The VSV infected CHO 15B cells were treated with cycloheximide (a protein
synthesis inhibitor).
1
• The cells were taken at different time points post the treatment and Golgi Fraction
was isolated.
2
• The Donor activity of the isolated fractions was tested by co-incubation this fraction
with Golgi fractions from uninfected wildtype cells and tested for radiation count.
3
 These data show that it
is a recently synthesized
pool of G protein in the
Golgi fraction of the 15B
cell membranes that is
subjected to transport to
receive GlcNac
 This pool is depleted
with a half time of about
10 min
DEPENDENCE OF RATE OF TRANSPORT ON
CYTOSOL CONCENTRATION
• Golgi fraction from VSV infected CHO 15B was isolated and co-
incubated with wild type CHO cells .
1
• Several such Assays were performed with varying cytosol
concentration.
2
• The GlcNAc incorporation was detected as a function of the amount
of cytosol added at various time intervals.
3
CONTD.
 It was demonstrated that the rate of transport is
saturable and is a function of the amount of cytosolic
proteins present.
DISCUSSION
• The authors describe a more direct assay for measuring transport of VSV G Protein
between successive compartments of the Golgi via complex incorporation of GlcNAc,
compared to their previous ones.
• ADVANTAGES:
 Utilises highly purified Golgi fractions.
 Negligible background radioactivity.
 More quantitative and rapid..
• Disadvantages:
 only measure G Protein molecules that have already arrived at Golgi: Origin of the
membrane bound vessivce – unknown.
CONTD.
• Evidences that old and new assay measure same:

 Basic design is same and conditions can be compared.

 Extensive copurification of donor activity Golgi complex.
 Prompt response to cycloheximide.