Question 1. MAB1 is a liver protein of unknown function. In the figure below mice have hadtheir MAB1 gene ”knocked out” (KO) making it impossible to produce MAB1 proteins in theirliver cells. MAB1 is normally only expressed in liver cells. Wild-type (WT) mice have not hadthis gene knocked out and express the MAB1 protein. HepG2 is a liver cell line that normallyexpresses the MAB1 protein. In the figure below the HepG2 cells have been transfected withsmall interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA.The siRNA used was either one directed towards MAB1 mRNAs or towards a control vWFmRNAs.

Examine the figure below

Albumin secretion from liver cells under altered MAB1 level conditions

A,
(upper panel

bottom panelpn

B,

Green fluorescence indicates how much siRNA entered into the cell during thetransfection. Red fluorescent indicates how much MAB1 protein is expressed in the cell 1 day afterthe siRNA transfection

Albumin secretion levels in blood plasma of MAB1 Knock Out (KO) and Wild Type (WT) pups

secretion) (

) (**,

C,

A. Data General outcomes shown:

1)2)

3)4)

Scale bars,

p

< 0.001; KO

n

= 25,WT

n

= 30). HepG2 liver cells treated with MAB-siRNA

compared with control cells treated with control vWF siRNA (vWF has no known role in cell

) (**,

< 0.001;

= 10).

immunofluorescent staining of MAB1 in HepG2 liver cells transfected with MAB1-siRNA or

vWF-siRNA-control.

Western blotting of HepG2 cells showing MAB1 protein level after the cells were treated with

MAB1-siRNA or treated with control vWF-siRNA.

20 μm. Tubulin is the loading control.

Explain what the data in section A above shows about MAB1 for ….a) WT vs. KO

Explain what the data in section A above shows about MAB1 for… b) vWF-siRNA treated

vs. MAB1 siRNA treated

What is siRNA? How does it perform its function in a cell? Why was it used in section B in

the above figure? What were the researchers trying to show?

Why was this western blot in section C done by the researchers? Why is it an important piece

of supporting evidence to the experiments done in parts (A) lower panel.

5) Collectively, what does all the data in the figure suggest about MAB1’s functional role in livercells.

B. MAB-1 potential functions:

1. If MAB-1 is a COPII coat adaptor protein such as sec23 or sec24 that use the SAR-1-GTPinteractions (see Chapter 13) how would that explain the data in part A (upper panel) of the figureabove.
2. If MAB-1 was a vesicular lectin found on the surface of trans-Golgi-derived exocytotic vesicles.And these lectins typically target Phosphatidylinositol 4,5 –PI (4,5) P2 membrane lipids (seechapter 13) how would that explain the data in part A (lower panel-siRNA treated) of the figureabove.
3. If MAB-1 is an ER resident albumin cargo receptor in the ER membrane (see chapter 13) with itsown KDEL sequence and we mutated the KDEL sequence, what would be the short and long-termeffects on plasma levels of secreted albumin in such a mutant animal.