Tryptophan‐rich basic protein (WRB) mediates insertion of the tail‐anchored protein otoferlin and is required for hair cell exocytosis and hearing

Abstract

The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophanrich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca 2+ channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.

Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo

“…To investigate the role of the TRC40 pathway in tissue physiology, we employed a previously established mouse line with loxP recombination sites introduced into the WRB allele26 allowing tissue-specific knockouts upon crossing with mouse lines expressing the Cre recombinase under the control of a promoter of interest. We created tissue-specific knockout models using Cre lines leading to WRB knockout in adult cardiomyocytes or hepatocytes.…”

“…Generation of conditional knockouts restricted to specific cell types allows the identification of relevant functions of components of the TRC40 pathway in tissue development and physiology. A recent study revealed that pancreatic beta cells lacking TRC40 show impaired insulin secretion23, whereas CAML is required for thymocyte development24, and loss of either receptor subunit in inner ear cells causes deafness2526.…”

“…Similarly, loss of WRB in Xenopus laevis has a strong impact on cardiac morphology and secretion of basement membrane matrix30. Other studies have identified an essential function of WRB in the formation of synaptic structures in photoreceptors and in inner ear cells263132. Due to the physiological focus on the analysis of the phenotypes, i.e.…”

Loss of GET pathway orthologs in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance

“…Conversely, lack of the mammalian GET3 orthologs TRC40 (transmembrane domain recognition complex of 40 kDa) leads to embryo lethality in mice, complicating global physiological analyses (21). Nevertheless, a handful of recent studies have started to analyze individual physiological consequences of the GET pathway in vivo using tissue-specific knockout (KO) approaches and observed that its function is required for a diverse range of physiological processes, such as insulin secretion, auditory perception, and photoreceptor function, in animals (22)(23)(24). A high degree of evolutionary conservation is often assumed, and it has been recognized that some components of the GET pathway are present in Arabidopsis thaliana (25,26).…”

New insights into cochlear sound encoding

“…Instead, hair cells employ the multi-C 2 -domain protein otoferlin 22 , a member of the ferlin family of membrane fusion-related proteins (reviewed in 23, 24), which is a tail-anchored protein and requires the TRC40 pathway for efficient targeting to the endoplasmic reticulum 25 . Otoferlin clusters below the synaptic ribbon 21 and seems to assume multiple roles in hair cell exocytosis.…”