Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin

Table 4 Fps1 yields following hyperosmotic shock

Osmotic shock	Time post-shock (min)	Fps1 yield relative to control	M:P ratio
No shock (2× CBS)	15	1.00 (0.61)	0.24 (0.00)
	30	0.59 (0.17)	0.22 (0.03)
	60	0.90 (0.48)	0.14 (0.01)
	90	1.21 (0.63)	0.10 (0.00)
Hyperosmotic shock (2× CBS, 0.8 M NaCl)	15	3.03 (0.21)*	1.08 (0.14)**
	30	2.62 (0.83)	1.24 (0.16)***
	60	1.73 (0.29)	0.29 (0.03)
	90	2.08 (0.58)	0.21 (0.02)*

Wild-type yeast cells were transformed with a plasmid expressing 5′∆1-43-FPS1-HA₃; uORF2 was therefore present (Table 1). Single transformants were cultured in shake flasks in 2 × CBS lacking histidine (to maintain the plasmid). At A₆₀₀ ~ 4, cultures were divided into two, harvested by centrifugation and the pellets suspended in either 2× CBS (no shock) or 2× CBS supplemented with 0.8 M NaCl (hyperosmotic shock). Fps1 yields were determined by immunoblot and analyzed using ImageJ [6] after 15, 30, 60 and 90 min; values are reported relative to control conditions (wild-type cells after 15 min in 2× CBS). All data shown are the mean of biologically-independent, triplicate determinations; where relevant, the standard error of the mean (SEM) is shown in parentheses. A two-tailed paired t test was used to compare Fps1 yields or polysome ratios of shocked cells with the corresponding values for control cells at the equivalent time point
* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001

ISSN: 1475-2859