Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding.

Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1(I1061T), is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1(I1061T) mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1(I1061T) mutation. Despite similar NPC1 mRNA levels between wild type and NPC1(I1061T) fibroblasts, NPC1 protein levels are decreased by 85% in NPC1(I1061T) cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1(I1061T) protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t((1/2)) 6.5 h) versus wild type Endo H-resistant species (t((1/2)) 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1(I1061T) protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1(I1061T) in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1(I1061T) protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1(I1061T) mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1(I1061T) mutation.

The generation and analysis of deficiencies within a small genomic region on the X chromosome of Drosophila melanogaster containing two genes, enhancer of rudimentary and CG15352.

The enhancer of rudimentary gene, e(r), encodes a 104-amino-acid, highly conserved transcription cofactor. Hypomorphic mutations of e(r) show an enhancement of a hypomorphic rudimentary mutant wing phenotype. These mutants in a wild-type background are viable, fertile, and morphologically wild-type. Since the only mutant alleles were hypomorphic, it was important to isolate null mutations to determine if any other phenotypes might be associated with a loss-of-function of e(r). We utilized a marked P element, P{SUPor-P, y(+)}, located 895 bp upstream of the start of transcription of e(r) to generate nineteen deficiencies in the region. Deficiencies of e(r) enhance the mutant wing phenotype of a hypomorphic rudimentary allele, r(hd1). In a wild-type background, the deficiencies of e(r), unlike the hypomorphic alleles, have a low viability and females have low fertility. The expression of e(r) in the nurse cells of the ovary is consistent with the low fertility, and suggests an ovarian function for e(r). Deficiencies of CG15352, the gene directly upstream of e(r), are not associated with any obvious mutant phenotypes and present the possibility that it encodes a nonvital or redundant function.

Regulation of the Drosophila melanogaster protein, enhancer of rudimentary, by casein kinase II.

The Drosophila melanogaster gene enhancer of rudimentary, e(r), encodes a conserved protein, ER. Most ER homologs share two casein kinase II (CKII) target sites. In D. melanogaster, these sites are T18 and S24. A third CKII site, T63, has been seen only in drosophilids. The conservation of these CKII sites, particularly T18 and S24, suggests a role for these residues in the function of the protein. To test this hypothesis, these positions were mutated either to alanine as a nonphosphorylated mimic or to glutamic acid as a phosphorylated mimic. The mutations were tested individually or in double or triple combinations for their ability to rescue either a wing truncation characteristic of the genotype e(r)(p1) r(hd1-12) or the synthetic lethal interaction between e(r)(p2) and the Notch allele N(nd-p). All of the substitutions as single mutations rescued both mutant phenotypes, arguing that individually the phosphorylation of the three residues does not affect ER activity. The double mutants T18A-S24A and T18E-S24E and the triple mutants T18A-S24A-T63A and T18E-S24E-T63E failed to rescue. Together the data support the following model for the regulation of ER by CKII. ER that is unphosphorylated at both T18A and S24 is inactive. CKII activates ER by phosphorylating either T18 or S24. Further phosphorylation to produce the doubly phosphorylated protein inactivates ER.