pH sensitive GFP mutant
The intracellular pH plays a decisive role in the life of cells. In my
project, I have tried to measure the internal pH of the Endoplasmic Reticulum
(ER), a cell organelle involved in protein synthesis and the response
of cells to external signals. For this project I used the more simple
slime mould Dictyostelium discoideum, as complex processes can best be
understood using simple organisms.
Using the methods of genetic engineering, I constructed a gene, which
codes for a fusion protein of pHluorin and calreticulin. pHluorin is
a pH sensitive mutant of the green fluorescent protein GFP. The pHluorin
spectra give us information about the pH of the protein’s environment.
Calreticulin is a resisdent protein of the ER and thus cannot be found
anywhere else inside the cell. When the two proteins are fused with each
other we get a pH indicator, which can be found in the ER only – we can
thus measure the specific internal pH of the ER. I found that the ER
is acid compared to the cytosol.
At this point we do not know whether the acid pH has more to do with
the role of the ER in protein synthesis or intracellular signalling.
This must be established through studies of the ER pH under various conditions
– something that is made possible by my work. These results will hopefully
help understand the reactions, which form the basis of life. |
