Heterotrimeric G protein signaling in the Arabidopsis unfolded protein response

Abstract

We present evidence that heterotrimeric G protein signaling is involved in cell death associated with the unfolded protein response (UPR) in Arabidopsis. Seedlings of homozygous agb1-2 (Gbeta-null mutation) mutant plants are markedly more resistant to growth inhibition by the protein glycosylation inhibitor tunicamycin (Tm) than either wild-type plants or gpa1-4 (Galpha-null mutation) mutants. Leaves of older Gbeta mutant plants show much less cell death when infiltrated with Tm than leaves of wild-type plants. The transcriptional response of Gbeta mutant plants to Tm is less pronounced than that of wild-type plants, as is the accumulation of BiP chaperone proteins. A majority of the Arabidopsis Gbeta protein is associated with the endoplasmic reticulum (ER) and cofractionates with membrane-associated ER luminal BiP. Consistent with its ER localization, Gbeta protein is degraded during the UPR, whereas Galpha protein is not. Taken together, these observations imply that the Gbeta protein, which forms a stable heterodimer with the Ggamma subunit, is involved in the signaling events that trigger UPR-associated cell death. The different Tm sensitivities of Galpha and Gbeta mutants, the ER localization of Gbeta, and the differential stabilities of Galpha and Gbeta proteins during the UPR suggest that the Gbetagamma complex serves a signaling function in the ER independent of its function in the Galphabetagamma heterotrimer.

Fig. 1.

Plants homozygous for null mutations in genes coding for the α and β subunits of the heterotrimeric G protein exhibit different sensitivities to Tm-induced growth inhibition. Homozygous gpa1-4 (Gα-null mutation), agb1-2 (Gβ-null mutation), and gpa1-4/agb1-2 double mutants were germinated on agar medium containing 0.3 μg/ml Tm for 6 days and then transferred to medium without Tm, allowed to recover for 10 days, and photographed.

Fig. 2.

Leaf senescence and cell death in wild-type and agb1-2 mutant plants. A 15 μg/ml Tm solution in 1.6% DMSO was infiltrated into leaves as described in Materials and Methods. Plants were photographed at 6 days after infiltration (A), and leaves were photographed at 8 days (B Left) or cleared and stained with trypan blue (B Right); leaves were harvested for determination of chlorophyll content at 8 days (C) or daily (D) to determine electrolyte release as described in Materials and Methods.

Fig. 3.

The Tm-induced UPR is attenuated in agb1-2 mutant plants. (A) Northern blot analyses of Col-0 and agb1-2 mutant plants treated with 15 μg/ml Tm. Total RNA was extracted at the indicated times, and 10 μg of total RNA was loaded into each lane. UPR marker probes are indicated on the right. 25S rRNA was used as an internal loading control. (B) Western blot analysis of Col-0 and agb1-2 mutant plants treated as in A. Total protein was extracted at the indicated times, and 30 μg of protein was loaded into each lane. Antibodies against AtBiP1 and 2 were used to probe the membrane.

Fig. 4.

Gβ protein colocalizes with BiP in the ER and is degraded during the UPR. (A) Aliquots of total cell extracts (T) and soluble (S) supernatant and pelleted microsomal (M) fractions as well the upper plasma membrane (Pm) fraction and lower intracellular membrane (Im) fractions after aqueous two-phase partitioning were analyzed together by PAGE and Western blotting using anti-GPA antibodies to detect Gα protein, anti-CFP antibodies to detect the CFP-Gβ fusion protein in extracts of transgenic plants expressing a Gβ-CFP fusion protein from the native Gβ promoter, and anti-BiP antibodies for the ER markers BiP1 and 2. (B) The Im fraction was sedimented through a 10–50% sucrose density gradient, and aliquots of each fraction were analyzed by PAGE and Western blotting as in A. (C) Gβ and Gα proteins were detected as in A in total extracts from Gβ-CFP transgenic plants injected with Tm at 30 μg/ml and sampled after 1–4 days. A comparable protein extract from agb1-2 mutant plants was used in the last lane and shows that the Gα protein is expressed in the absence of the Gβ protein. (D) Band intensities from C are expressed as a percentage of control values.