GORAB is a protein that localises to the trans-Golgi network (TGN) and is known to interact with Rab6. The loss of GORAB leads to gerodermia osteodysplastica (GO), an autosomal recessive disorder which results in lax skin, precocious skin aging, osteoporosis, susceptibility to fractures and joint hyperelasticity. Both the function of GORAB and the mechanism of pathogenesis in GO patients are poorly defined. In this study, the cellular functions of GORAB have been investigated. Using a variety of approaches (yeast two-hybrid assays, pull-downs with recombinant proteins, proximity biotinylation assays combined with mass spectrometry and co-immunoprecipitations) it was possible to establish a network of interactions with Golgi-localised proteins, including Arf GTPases and the COPI-associated protein Scyl1, that suggests a possible role of GORAB in COPI-mediated trafficking. Consistent with this hypothesis, ultrastructural changes of the Golgi apparatus were observed as were abnormal protein glycosylation in primary skin fibroblasts derived from GO patients, detected using both lectin binding assays and mass spectrometric glycan profiling. Moreover, immuno-electron microscopy studies revealed unequal distribution of GORAB within the TGN and fluorescence recovery after photobleaching experiments show GORAB being very stably associated with Golgi membranes. These properties of GORAB seem to result from its ability to oligomerise and to interact with vimentin filaments. Based on these data, a model of GORAB acting as a scaffolding protein that organises sites of COPI budding at the TGN has been proposed. Additionally, analysis of both published and newly identified GORAB mutations found in GO patients revealed that they affect various properties of GORAB including its interaction with small GTPases and GORAB ability to oligomerise, which suggests that these features are important for GORAB cellular functions. Together, these data suggest that the underlying cause of the skin and bone defects observed in GO patients is impaired COPI trafficking at the Golgi apparatus resulting in abnormal glycosylation of extracellular matrix proteins.

Planar cell polarity (PCP) refers to the coordinated orientation, movement, or structure of cells within the plane of a tissue. Zebrafish PCP mutants such as the vangl2 mutant exhibit defects in convergent extension, neural tube morphogenesis, and ciliary positioning. Tmem14a is a putative tetraspanin protein that was identified as an potential interactor of Vangl2 in a membrane yeast-two hybrid screen. GFP-tagged versions of Tmem14a are localized to the trans-Golgi network in zebrafish neuroepithelial cells. Knockdown of Tmem14a activity results in convergent extension defects, an ectopic accumulation of cells in the neural tube, and disorganized cilia. The localization of GFP-tagged Tmem14a to the trans-Golgi network suggested that Tmem14a plays a role in the trafficking of core PCP components to the cell membrane. Indeed, the membrane localization of GFP-Vangl2 was disrupted in Tmem14a morphants. Thus, Tmem14a is an interactor of Vangl2 and a novel regulator of vertebrate planar cell polarity signaling.

Planar cell polarity (PCP) refers to the coordinated orientation, movement, or structure of cells within the plane of a tissue. Zebrafish PCP mutants such as the vangl2 mutant exhibit defects in convergent extension, neural tube morphogenesis, and ciliary positioning. Tmem14a is a putative tetraspanin protein that was identified as an potential interactor of Vangl2 in a membrane yeast-two hybrid screen. GFP-tagged versions of Tmem14a are localized to the trans-Golgi network in zebrafish neuroepithelial cells. Knockdown of Tmem14a activity results in convergent extension defects, an ectopic accumulation of cells in the neural tube, and disorganized cilia. The localization of GFP-tagged Tmem14a to the trans-Golgi network suggested that Tmem14a plays a role in the trafficking of core PCP components to the cell membrane. Indeed, the membrane localization of GFP-Vangl2 was disrupted in Tmem14a morphants. Thus, Tmem14a is an interactor of Vangl2 and a novel regulator of vertebrate planar cell polarity signaling.