The Saccharomyces cerevisiae SOP1 andSOP2 Genes, Which Act in Cation Homeostasis, Can Be Functionally Substituted by the Drosophila lethal(2)giant larvae Tumor Suppressor Gene

Abstract

By complementation of a salt-sensitive mutant of Saccharomyces cerevisiae, we cloned the SOP1 gene, encoding a 114.5-kDa protein of 1033 amino acids. Cells deleted for SOP1 exhibited sensitivity to sodium stress, but showed no sensitivity to general osmotic stress. Following exposure of sop1⌬ cells to NaCl stress, the intracellular Na ؉ level and the Na ؉ /K ؉ ratio rose to values significantly higher than in wild type cells. Deletion of SOP2, encoding a protein sharing 54% amino acid identity with Sop1p, produced only slight Na ؉ sensitivity. Cells carrying a sop1⌬sop2⌬ double deletion became, however, hypersensitive to Na ؉ and exhibited increased sensitivity also to Li ؉ and K ؉ , suggesting involvement of both SOP1 and SOP2 in cation homeostasis. The predicted amino acid sequences of Sop1p and Sop2p show significant homologies with the cytoskeletal-associated protein encoded by the Drosophila lethal(2)giant larvae tumor suppressor gene. Immunolocalization of Sop1p revealed a cytoplasmic distribution and cell fractionation studies showed that a significant fraction of Sop1p was recovered in a sedimentable fraction of the cytosolic material. Expression of a Drosophila l(2)gl cDNA in the sop1⌬sop2⌬ strain partially restored the Na ؉ tolerance of the cells, indicating a functional relationship between the Sop proteins and the tumor suppressor protein, and a novel function in cell homeostasis for this family of proteins extending from yeast to human.

Yeast Lacking theSRO7/SOP1-encoded Tumor Suppressor Homologue Show Increased Susceptibility to Apoptosis-like Cell Death on Exposure to NaCl Stress

“…Already, a 10-min exposure to 0.7 M NaCl killed ϳ30% of either population ( Figure 1A), and Ͻ5% remained viable after 6 h. Equivalent treatment of wild-type cells resulted in Ͻ5% loss in viability. These results demonstrate that sro7⌬ and sro7⌬sro77⌬ mutants not only exhibit reduced ability to grow at moderate salt stress, as has been reported previously (Larsson et al, 1998) but also that the cells are actually killed by the stress.The observations prompted us to analyze the cells for the presence of ROS, which are considered a key stimulus for celldeathbyapoptosis (Madeoetal.,1999).Usingdihydrorhodamine 123 as ROS indicator, accumulation of ROS was observed in ϳ60% of sro7⌬ or sro7⌬sro77⌬ mutants exposed to 0.8 M NaCl (Figure 1, C and D). Control wild-type cells showed little dihydrorhodamine staining, even when exposed to 1 M NaCl ( Figure 1D).…”

“…Instead, Ena1p is delivered to the vacuole where it is degraded. Deletion of SRO7 together with its iso-gene SRO77 results in hypersensitivity to NaCl stress (Larsson et al, 1998), and a coldsensitive phenotype (Lehman et al, 1999). At nonpermissive temperature, the sro7⌬sro77⌬ mutant shows defective secretion and accumulation of post-Golgi vesicles (Lehman et al, 1999).…”

“…However, overexpression of Bcl-2 did not suppress the salt-sensitive phenotype of a yeast hog1⌬ mutant, which has decreased capacity to osmoregulate, but seems to maintain ion homeostasis. We have observed that deletion of the SOP1/ SRO7 gene (hereafter referred to as SRO7) brings about increased sensitivity to NaCl stress and a defective intracellular ion homeostasis (Larsson et al, 1998). SRO7 is a yeast homologue of the Drosophila l(2)gl tumor suppressor gene.…”

“…Rich YPD medium (2% glucose) was used for growth and survival assays, whereas minimal YNB media (2% glucose) plus essential amino acids were used for selective growth. For salt stress conditions, YPD containing 2 M NaCl was added to yeast cultures to obtain final NaCl concentrations.The yca1⌬sro7⌬ mutant was constructed using the sro7::LEU2 deletion cassette in pBluescriptKS ϩ (Larsson et al, 1998). The plasmid was cut with restriction enzymes HindIII and Notl (Roche Diagnostics, Mannheim, Germany) and the ycal⌬ mutant transformed with the sro7::LEU2 fragment of 3.5-kb subsequent to gel purification (QIAquick gel extraction kit; QIAGEN, Hilden, Germany).…”

The human protein Hugl-1 substitutes for Drosophila Lethal giant larvae tumour suppressor function in vivo

“…Recently, a complex of PAR-6 and atypical protein kinase C was shown to bind and phosphorylate mammalian Lgl (Plant et al, 2003;Yamanaka et al, 2003). Both mammalian and Drosophila proteins are able to rescue yeast strains double deficient for yeast Lgl homologues, Sop1 and Sop2 (Larsson et al, 1998;Kim et al, 2003). While the role of Lgl protein in cell proliferation control has been assessed in Drosophila, the question must still be addressed for the human counterpart.…”