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Tutorial:
BIOL200 Tutorial 7
Unit 4 Problem Solving Worksheet
Problem 4.3.5 (tags: #Golgi processing, #differential centrifugation, #mutant analysis, #radiolabelling,
#experimental controls, #prediction, #animals)

Over the years, much work has been done by scientists to determine how cargo moves through the cisternae of the
Golgi Apparatus. Many models have been put forward to explain. The Cisternal Maturation Model was one of the
first models in the 1960's. It was based on morphological observations of scale synthesis in a number of different
types of photosynthetic algal protists. By the 1990s, the generally accepted model was called the Vesicle
Transport Model. This was based on some quite clever experiments with animal cells. Drawings to represent these
2 models are shown below:

In the Cisternal Maturation Model (Panel A), new cisternae form continuously as vesicles from the ER coalesce at
the cis face of the Golgi. Each newly formed cisterna moves through the stack with appropriate modification of its
contents, and finally breaks up into transport vesicles at the trans face. In the Vesicle Transport Model (Panel B),
cisternae remain fixed and the maturing glycoproteins move from the cis to the trans cisternae in transport
vesicles

One experimental test of these models involved following the path of a protein (called protein A) through the
Golgi apparatus. As it moves through the Golgi, Protein A gets glycosylated, just like any other protein. It is
normally glycosylated with both:

 Glucose (in the medial compartment of the Golgi) and…

 Galactose (in the trans compartment of the Golgi).

The experiment made use of mutant cells that are defective in the addition of galactose to glycoproteins, and
normal cells that had no problem adding galactose to glycoproteins.
In each experiment, some Golgi were labeled with radioactive glucose. The cells containing these labeled Golgi
were then fused with cells with unlabeled Golgi. As such the hybrid cell that was produced contained functional
Golgi of both types (with and without labeled glucose). (For more information, see Rothman et al (1984). J Cell
Biol 99: 260-271, or Balch et al. (1984). Cell 39: 405-406).
After an hour, the cells were dissolved in detergent and the protein being studied was isolated via centrifugation.
This protein was separated into two fractions, molecules with attached galactose and molecules without galactose.
The fraction with attached galactose was precipitated (pellet), leaving the rest of the protein in solution
(supernatant).
The following distribution of label was observed in control and experimental cell combinations:

Fraction of label in
Fraction of label in pellet
Cell combination supernatant (protein
(protein with galactose)
without galactose)
Labeled wild-type cells fused with non-
85% 15%
labeled wild-type cells

Labeled mutant cells fused to non-labeled

5% 95%
mutant cells
Labeled mutant cells fused with non-
45% 55%
labeled wild-type cells
Based on the information and data above, see if you can answer the following questions (you’ll need to make sure
that you fully understand what was done before you’ll be able to answer these questions):

a) This experiment is testing for movement of proteins between what two Golgi compartments? Explain your
answer briefly.
They are testing the movement of proteins between medial and trans Golgi. This is done by observing if
galactose is able to be added to the protein. Mutant cells do not have the enzyme to add galactose in the trans
compartment, while the wildtype does. By fusing the two types of cells (so having both mutant and wildtype
trans compartments) they can test which model is in line with the results.

b) If proteins moved through the Golgi apparatus by cisternal maturation, what result would you predict for row
3 of the table above? Why?
In the cisternal maturation model, the protein stays in the mutant medial compartment, which then becomes a
trans compartment that still does not have the enzyme to add galactose to the protein. The labeled protein
won’t be able to reach a wildtype trans compartment, so 0% of the proteins will be in pellet and 100% are in
supernatant (all do not have galactose added)

c) If proteins moved through the Golgi apparatus by vesicle transport, what result would you predict for row 3 of
the table above? Why?
In the vesicle transport model, the protein moves between medial and trans compartments through vesicles.
The labeled protein in a vesicle has a 50% chance of entering a mutant trans compartment (therefore no
galactose added), and a 50% chance of entering a wildtype trans compartment (therefore galactose added).
Thus, 50% of the labeled proteins are in pellet, and 50% are in supernatant.

d) Explain which model is supported by the results in the table? Why?

The results support the vesicle transport model, as 45% of protein are found in pellet (approximately half), and
the other half are in supernatant.

e) What false assumption do we now know that the researchers made that make their results inconclusive?
Explain why this is problematic for the experimental design.
This experiment assumed that the enzymes in each compartment moves along and stays in the cisternal
maturation model. However, when medial Golgi mature into trans Golgi, the medial enzymes move from the
new trans Golgi back to a new medial Golgi through retrograde transport. This means that in fused cells,
wildtype enzymes in the trans compartment may be brought back to the mutant medial compartment, so
galactose may be able to be added. This makes the prediction in part b) incorrect, so the results may be due to
either of the models.