Fig 7.

Glucose depletion triggers dephosphorylation of Nrd1 and its relocalization to nuclear speckles. (A) Nrd1HA is dephosphorylated upon glucose depletion. NRD1HA cells were transferred from SC with glucose at 25°C to SC lacking glucose at 25°C. Total protein extracts collected at 20-min intervals were analyzed by Western blotting using antibodies to Nrd1 and to Pgk1, which served as a loading control. (B to I) Starvation-induced formation of nuclear speckles containing Nrd1. Nrd1-GFP (B [green], C, F [green], and G) does not substantially colocalize with the nucleolar marker Sik1-RFP (B [red], D, F [red], and H) in either fed (B to E) or glucose-deprived (F to I) cells. Nrd1-GFP is dispersed within the nucleus (Hoechst 33342) (B [blue], E, F [blue], and I) in fed cells but forms punctae in or around the nucleus in glucose-depleted cells. (J to O) Nrd1-GFP (J [green], K, M [green], and N) appears to colocalize with Nab3-mCherry (J [red], L, M [red], and O) in fed (J to L) and glucose-deprived (M to O) cells. (P to U) Nrd1-GFP (P [green], Q, S [green], and T) does not substantially colocalize with the P-body marker Dcp2(1-300)-RFP (P [red], R, S [red], and U) in fed (P to R) or glucose-deprived (S to U) cells. (V to AA) Nrd1-GFP (V [green], W, Y [green], and Z) does not substantially colocalize with the stress granule marker Pub1-mCherry (V [red], X, Y [red], and AA) in fed (V to X) or glucose-deprived (Y to AA) cells. Log-phase cells were washed and incubated in SC medium with glucose (+Glc) or without glucose (−Glc) prior to visualization.