Extensive degradation of mutant-type D123 protein is responsible for temperature-sensitive proliferation inhibition in 3Y1tsD123 cells

A temperature-sensitive mutant of 3Y1, 3Y1tsD123, reversibly arrested in G1 phase of cell cycle at the restrictive temperature of 39.8 degrees C, show s a single amino acid exchange in the D123 protein. In this study, we found that the D123 protein level in 3Y1tsD123, which was 1/8 of that in 3Y1 com pared at the permissive temperature of 33.9 degrees C, lowered to 1/4 after a shift to the restrictive temperature. During inhibition of protein synth esis with cycloheximide, the D123 protein level in 3Y1tsD123 decreased mark edly depending on the incubation temperature, compared with that in 3Y1, in dicating that the lowered levels of D123 protein in 3Y1tsD123 are due to it s degradation. Unexpectedly, 2 stably temperature-resistant clones were iso lated after transfection of SV-3Y1tsD123 (SV40-transformed 3Y1tsD123, which shows cell death instead of G1 arrest at the restrictive temperature) with the cDNA of the mutant-type (3Y1tsD123-derived) D123 protein, The D123 pro tein in both clones degraded extensively at both temperatures, suggesting t hat the overexpression of the mutant-type D123 protein exceeds its degradat ion. Both temperature-resistant clones contained higher levels of D123 prot ein at the restrictive temperature than did SV-3Y1tsD123 at the permissive temperature. We concluded that the lowered D123 protein level at the restri ctive temperature induces the temperature-sensitive characteristics of 3Y1t sD123 and SV-3Y1tsD123.