Nuclear-Gene Mutations Suppress a Defect in the Expression of the Chloroplast-Encoded Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase1

The green alga Chlamydomonas reinhardtii mutant 76–5EN lacks photosynthesis because of a nuclear-gene mutation that specifically inhibits expression of the chloroplast gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Photosynthesis-competent revertants were selected from mutant 76–5EN to explore the possibility of increasing Rubisco expression. Genetic analysis of 10 revertants revealed that most arose from suppressor mutations in nuclear genes distinct from the original 76–5EN mutant gene. The revertant strains have regained various levels of Rubisco holoenzyme, but none of the suppressor mutations increased Rubisco expression above the wild-type level in either the presence or absence of the 76–5EN mutation. One suppressor mutation, S107–4B, caused a temperature-conditional, photosynthesis-deficient phenotype in the absence of the original 76–5EN mutation. The S107–4B strain was unable to grow photosynthetically at 35°C, but it expressed a substantial level of Rubisco holoenzyme. Whereas the 76–5EN gene encodes a nuclear factor that appears to be required for the transcription of the Rubisco large-subunit gene, the S107–4B nuclear gene may be required for the expression of other chloroplast genes.

Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.