> I am interested in methods to enlarge yeast cells for patch clamp
>and (possibility) intracellular measurements of ion transport activity. I
>recently read in The Life of Yeasts (Phaff, Miller and Mrak) that
>tunicamycin causes Saccharomyces cerevisiae to enlarge and become
>spherical, without significantly impairing growth. Unfortunately, thie
>wasn't referenced, and the only references I could uncover examined the
>effects of tunicamycin on yeast protoplasts.
> Has anyone worked on this? I would be very interested in how large
>the cells become. Alternatively, could someone point me in the direction
>of any references to this effect of tunicamycin?
>Any help appreciated.............
> (York University - Biology)
> (e-mail: fs300541 at sol.yorku.ca)
I know tunicamycin works by specifically inhibiting the first gene in the
dolichol pathway of protein N-glycosylation (ALG7). It causes enlargement of
cells due to ongoing protein synthesis in the abscence of cell division. It
causes cell death after several generation times, since the ALG7 gene it
inhibits is essential. Cells arrest from cell cycle with a G2 DNA content.
Some references to consider:
Arnold, E. and Tanner, W. (1982) An obligatory role of protein
glycosylation in the life of yeast cells. FEBS Lett. 148, 49-53.
Vai, M., Popolo, L., and Alberghina, L. (1987) Effect of tunicamycin on cell
cycle progression in budding yeast. Experimental Cell Research 171:448-459.
Unfortunately, it is not known (yet) what specific effects altering a cell's
ability to N-glycosylate proteins has.....but you definitely ought to consider
that before you use tunicamycin in your studies. Some references for
N-glycosylation studies in yeast:
Kukuruzinska, M. and Lennon, K. (1994) Growth-related coordinate regulation of
the early N-glycosylation genes in yeast. Glycobiology 4:437-443.
Kukuruzinska, M. and Lennon, K. (1995) Diminished activity of the first
N-glycosylation enzyme, dolichol-P-dependent N-acetylglucosamine-1-P
transferase (GPT), gives rise to mutant phenotypes in yeast. Biochim Biophys
Pretel, R., Lennon, K., Bird, A., and Kukuruzinska, M. (1995) Expression of
the first N-glycosylation gene in the dolichol pathway, ALG7, is regulated at
two major control points in the G1 phase of the Saccharomyces cell cycle.
Experimental Cell Research 219:477-486.
Lennon, K., Pretel, R., Kesselheim, J. , te Heesen, S., and Kukuruzinska, M.A.
(1995) Proliferation-dependent differential regulation of the dolichol pathway
genes in Saccharomyces cerevisiae. Glycobiology 5:633-642.
Yes, I know it looks like I'm "blowing my own horn", but I just want to make a
point that tunicamycin treatment of your cells might really screw up the
interpretability of your results.
BU Med Center
Center for Advanced Biomedical Research