Lecture 14 - papers on glycans

Question 1

What is the signal required for selective export from the endoplasmic reticulum?

Answer 1

A di acidic signal (-Asp-X-Glu-)

Question 2

What is the Yxxφ motif used for?

Answer 2

It is a cell surface internalization signal.

Question 3

How was the VSV-G tracked at various stages of secretion?

Answer 3

GFP was attached to VSV-G glycoprotein to track it’s movement through the cell. A temperature sensitive mutant was used to trap it at various stages of secretion.

Question 4

What are the steps in analysing the ER export of glycoproteins from the ER.

Answer 4

1) cDNA coding VSV-G and GFP is added to a plasmid and is transfected into cultured cells.
2) Cells are labelled with S35 methionine. This is in addition to a temperature of 39 degrees so that proteins are retained within the ER.
3) Peptides are added which are being tested to see if they inhibit export from the ER. To enter the ER the cells need to be permeabilised.
4) Decrease temperature to permissive temperature and analyse the 35S methionine labelled VSV-G glycoprotein for sugars which are present due to modification within the golgi.
* test of step 4 can be done via use of endo H enzyme + immuno precipitate.

Question 5

What is the function of the diacidic motif?

Answer 5

Diacidic motif is important in making the export of glycoprotein from the ER more efficient.

Question 6

Why is using mammalian cells for testing the export site properties of the ER a bad idea?

Answer 6

Mammalian cells have 100s of tER locations. Yeast have much less (specifically Pichia Pastoris ) and so yeast are a better organism to use.

Question 7

What causes certain proteins to be named Sec proteins?

Answer 7

Sec refers to a bunch of mutants initially pulled out of yeast cells.

Question 8

What are the steps of identifying the properties of the eER sites?

Answer 8

1) tER sites are marked with GFP by mutating the Sec13 (outer coat of COP2) subunits. Now Sec13 can be seen and marks tER sites.
2) Sec12-GG contains another tag on it.
3) Mutate the yeast cells with the tags.
4) Screen mutants for altered staining pattern of Sec13-GFP. Screening can be used to isolate conditional mutants such as temperature sensitive mutants.
5) High temperature should cause accumulation of sec proteins.
6) Wild type genome is screened and compared to the genome of the mutated yeast. Interactions between mutations are identified in this steps.