Figure 1.

Overexpression of cnx1⁺ induces cell death. (A) Schematic representation of S. pombe calnexin, Cnx1p. SP corresponds to the signal peptide (22 amino acids; aa), followed by the N-terminal part (98 aa; symbolized by the forward-hatched box). The highly conserved central domain (hcd) is represented by a white box (317 aa). The C-terminal part of lumenal domain of calnexin is 52-aa long and is represented by the backward-hatched box. The transmembrane domain (TM, black box) and the cytosolic tail (punctuated box) span 23 and 48 aa, respectively. (B) Survival of cells overexpressing Cnx1p (strain SP8007R) and control strains (empty vector, SP7975R; Pdi1p, SP16058; and Sec61β, SP15302) was assayed by serial dilution on inducing plates (without thiamine) or repressing plates (with thiamine). Samples of 10 μl of four 10-fold serial dilutions (10⁻¹–10⁻⁴) of cells at 0.5 OD₅₉₅ were spotted on selective MM and incubated at 30°C for 7 d (see Materials and Methods). (C) Percentage of dead cells measured by staining with fluorescent vital dye Phloxin B. Cells overexpressing Cnx1p (strain SP8007R) and control strains (empty vector, SP7975R; Pdi1p, SP16058; and Sec61β, SP15302) were stained with Phloxin B after 48 h of induction of overexpression and fluorescent cells were quantified by FACS. Stained cells were considered as dead. A global ANOVA showed that the values obtained among the strains were significantly different (p = 0.000). In C, vertical black arrows symbolize overexpression of the indicated protein.