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Wolfram syndrome is caused by mutations in WFS1 encoding wolframin, a polytopic membrane protein of the endoplasmic reticulum. Here, we investigated the molecular pathomechanisms of four missense and two truncating mutations in WFS1. Expression in COS-7 cells as well as direct analysis of patient cells revealed that WFS1 mutations lead to drastically reduced steady-state levels of wolframin. All mutations resulted in highly unstable proteins which were delivered to proteasomal degradation. No wolframin aggregates were found in patient cells suggesting that Wolfram syndrome is not a disease of protein aggregation. Rather, WFS1 mutations cause loss-of-function by cellular depletion of wolframin.

Mutations of the WFS1 gene are responsible for Wolfram syndrome, a rare, recessive disorder characterized by early-onset, non-autoimmune diabetes mellitus, optic atrophy and further neurological and endocrinological abnormalities. The WFS1 gene encodes wolframin, a putative multispanning membrane glycoprotein of the endoplasmic reticulum. The function of wolframin is completely unknown. In order to characterize wolframin, we have generated polyclonal antibodies against both hydrophilic termini of the protein. Wolframin was found to be ubiquitously expressed with highest levels in brain, pancreas, heart and insulinoma beta-cell lines. Analysis of the structural features provides experimental evidence that wolframin contains nine transmembrane segments and is embedded in the membrane in an N(cyt)/C(lum) topology. Wolframin assembles into higher molecular weight complexes of approximately 400 kDa in the membrane. Pulse-chase experiments demonstrate that during maturation wolframin is N-glycosylated but lacks proteolytical processing. Moreover, N-glycosylation appears to be essential for the biogenesis and stability of wolframin. Here we investigate, for the first time, the molecular mechanisms that cause loss-of-function of wolframin in affected individuals. In patients harboring nonsense mutations complete absence of the mutated wolframin is caused by instability and rapid decay of WFS1 nonsense transcripts. In a patient carrying a compound heterozygous missense mutation, R629W, we found markedly reduced steady-state levels of wolframin. Pulse-chase experiments of mutant wolframin expressed in COS-7 cells indicated that the R629W mutation leads to instability and strongly reduced half-life of wolframin. Thus, the Wolfram syndrome in patients investigated here is caused by reduced protein dosage rather than dysfunction of the mutant wolframin.