Figure 5.

Genetic interactions between TLG and SNC genes in yeast. (A) Disruption of TLG1 or TLG2 in snc cells (JG8) slows cell growth and results in conditional lethality. Left panel: snc cells (JG8) transformed with a control plasmid (snc), a plasmid expressing SNC1 constitutively (SNC1), or snc tlg2 cells. Right panel: snc cells (JG8) transformed with a control plasmid (snc), a plasmid expressing SNC1 constitutively (SNC1), or snc tlg1 cells. All cells first were grown at 26°C on medium containing galactose, before replica plating onto medium containing glucose to deplete Snc1. After 24 h, patches were replica-plated onto the following: glucose-containing plates preequilibrated to 15°C, 26°C, 30°C, and 37°C; rich medium (YPD); and rich medium containing galactose (YPG). Plates were incubated for 30 h. (B) Growth curves of snc tlg cells on galactose- and glucose-containing medium. Left panel: snc (JG8), snc cells expressing SNC1, snc tlg1, and snc tlg2 cells were grown to log phase at 26°C on galactose-containing medium (to induce SNC1). Equal amounts of cells then were seeded into freshgalactose-containing medium (GAL), and optical density (measured at 600 nm) at 26°C was monitored as a function of time. Right panel: Wild-type (WT), snc (JG8), snc cells expressing SNC1, snc tlg1, and snc tlg2 cells were grown to log phase at 26°C on galactose-containing medium before transfer to glucose-containing medium for 24 h to deplete Snc1. Equal amounts of cells then were seeded into glucose-containing medium (GLU) and optical density (measured at 600 nm) at 26°C was monitored as a function of time. (C) snc tlg cells expressing SNC1^ala43 are not rescued for growth defects. Left panel: snc (JG8), snc cells expressing SNC1, snc tlg1, and snc tlg2 cells were grown to log phase at 26°C on galactose-containing medium (to induce SNC1). Equal amounts of cells then were seeded into fresh galactose-containing medium (GAL) and optical density (at 600 nm) at 26°C was monitored as a function of time. Right panel: WT, snc (JG8), snc cells expressing SNC1, SNC1^ala43 tlg1, and SNC1^ala43 tlg2 cells were grown to log phase at 26°C on galactose-containing medium before transfer to glucose-containing medium for 24 h to deplete Snc1. Equal amounts of cells then were seeded into fresh glucose-containing medium (GLU) and optical density (at 600 nm) at 26°C was monitored. (D) FM4–64 labeling of vacuoles is impaired in snc tlg and SNC1^ala43 tlg cells. snc (JG8), snc tlg1, and snc tlg2 cells were grown continually on galactose-containing medium at 26°C before labeling with FM4–64 (top three panels). snc (JG8), SNC1^ala43 tlg1, and SNC1^ala43 tlg2 cells were grown continually on glucose-containing medium at 26°C before labeling with FM4–64 (bottom three panels).