Poor fluorescence of GFP-fusion proteins in yeast

Shelley s.robison at auckland.ac.nz
Sun Apr 28 22:57:49 EST 2002


I'm having difficulty with transient expression of a C-terminal GFP
fusion using a membrane protein fused to S65T.  I am transforming
S.cerevisiae cells and selecting on appropriate media.  However,
following restreaking etc, visualisation of the yeast cells with
fluorescence microscopy reveals only a very small proportion of cells
that fluoresce with GFP.  These cells are usually very bright.  I have
found that the cells lines are able to functionally complement deletion
mutants lacking my gene of interest, indicating that the membrane
protein at least is being transcribed and translated.  However, because
I am interested in using the GFP-fusion for subcellular localisation
studies I need to show that the cells which fluoresce also complement
the deletion mutants correctly.  Does anyone know why I might be getting
such a low number of fluorescing cells and how this number could be
increased significantly?  I have tried variations in growth temperatures
and microscopy but to no avail so far.

Thanks in advance,



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