The Golgin GMAP-210

The protein GMAP-210 (Golgi Microtubule Associated Protein of 210 kDa) is a long coiled-coil protein, which localises to the Golgi apparatus. It is part of the loosely defined protein group of the golgins, which are involved in establishing the Golgi morphology and in vesicular trafficking around the Golgi. By using biochemical, cell biological and molecular biological methods GMAP-210 was examined in regards to its Golgi targeting capability, its interaction partners and its function in establishing Golgi morphology and positioning. In vitro and in vivo experiments showed that GMAP-210 targets to the Golgi via its C-terminal GRAB domain. Its proposed interaction with Arf1, however, could not be definitely determined, although there is strong evidence for it. Arf1 binding to the GRAB domain was hindered in the full-length protein, but not with short C-terminal fragments containing the minimal GRAB domain. This implies that additional factors are needed for GMAP-210 Golgi binding. A yeast 2-hybrid screen of the entire family of small Rab GTPases identified the Golgi and ER localised Rab1 as a novel interaction partner of GMAP-210. GMAP-210 also labels vesicular tubular structures in the cell, which partially overlap with COPII and ERGIC53, components of the early secretory pathway. This gives additional evidence that GMAP- 210 is involved in ER to Golgi transport. Trafficking of a model substrate, the vesicular stomatitis virus G-protein (VSV-G), however, was not impaired in the absence of GMAP- 210. This indicates that GMAP-210 functions only in specialised transport pathways. Knockdown of GMAP-210 in HeLa L cells by siRNA changed the Golgi morphology and the Golgi fragmented into a cluster of vesicles. Its overexpression caused the Golgi to grow long tubular structures. Both effects on morphology could only be observed in HeLa L cells, not in hTERT-RPE1 cells. As direct interaction with microtubules or γ-tubulin could not be detected, and GMAP-210 is therefore unlikely to affect Golgi morphology by directly perturbing microtubule function. GMAP-210 knockdown by siRNA also showed its interaction with the intraflagellar transport protein IFT20. This protein lost its Golgi localisation when GMAP-210 was depleted. Both proteins interacted directly. GMAP-210, however, was not involved in primary cilium formation in hTERT-RPE1 cells and loss of IFT20 from the Golgi did not impair formation of the cilium, proposing that the Golgi pool of IFT20 had a function apart from intraflagellar transport and formation of the primary cilium. These results set GMAP-210 apart from the archetypal golgins GM130 and p115 and indicate that GMAP-210 is involved in a highly specialised transport pathway, which could nevertheless influence the morphology of the Golgi apparatus in certain cell types

Functional dissection of Rab GTPases involved in primary cilium formation

Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125–148; Singla, V., and J.F. Reiter. 2006. Science. 313:629–633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517–524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia

Impaired proteoglycan glycosylation, elevated TGF-β signaling, and abnormal osteoblast differentiation as the basis for bone fragility in a mouse model for gerodermia osteodysplastica

<div><p>Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. <i>GORAB</i>, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the <i>Gorab</i>^<i>Null</i> full knockout, <i>Gorab</i> was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only <i>Gorab</i>^Prx1 and <i>Gorab</i>^Runx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of <i>Gorab</i>^<i>Null</i> mutants and in bone of <i>Gorab</i>^Prx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from <i>Gorab</i>^<i>Null</i> mutants. In bone from <i>Gorab</i>^Prx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured <i>GORAB</i>-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-β in <i>Gorab</i>^Prx1 bone tissue leading to enhanced downstream signalling, which was reproduced in <i>GORAB</i>-deficient fibroblasts. Our data suggest that the loss of <i>Gorab</i> primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.</p></div

Conceptual Design of a Hydrogen-Hybrid Dual-Fuel Regional Aircraft Retrofit

A wide range of aircraft propulsion technologies is being investigated in current research to reduce the environmental impact of commercial aviation. As the implementation of purely hydrogen-powered aircraft may encounter various challenges on the airport and vehicle side, combined hydrogen and kerosene energy sources may act as an enabler for the first operations with liquid hydrogen propulsion technologies. The presented studies describe the conceptual design of such a dual-fuel regional aircraft featuring a retrofit derived from the D328eco under development by Deutsche Aircraft. By electrically assisting the sustainable aviation fuel (SAF) burning conventional turboprop engines with the power of high-temperature polymer-electrolyte fuel cells, the powertrain architecture enables a reduction of SAF consumption. All aircraft were modeled and investigated using the Bauhaus Luftfahrt Aircraft Design Environment. A description of this design platform and the incorporated methods to model the hydrogen-hybrid powertrain is given. Special emphasis was laid on the implications of the hydrogen and SAF dual-fuel system design to be able to assess the potential benefits and drawbacks of various configurations with the required level of detail. Retrofit assumptions were applied, particularly retaining the maximum takeoff mass while reducing payload to account for the propulsion system mass increase. A fuel cell power allocation of 20% led to a substantial 12.9% SAF consumption decrease. Nonetheless, this enhancement necessitated an 18.1% payload reduction, accompanied by a 34.5% increment in propulsion system mass. Various additional studies were performed to assess the influence of the power split. Under the given assumptions, the design of such a retrofit was deemed viable

GORAB Missense Mutations Disrupt RAB6 and ARF5 Binding and Golgi Targeting

Gerodermia osteodysplastica is a hereditary segmental progeroid disorder affecting skin, connective tissues, and bone that is caused by loss-of-function mutations in GORAB. The golgin, RAB6-interacting (GORAB) protein localizes to the Golgi apparatus and interacts with the small GTPase RAB6. In this study, we used different approaches to shed more light on the recruitment of GORAB to this compartment. We show that GORAB best colocalizes with trans-Golgi markers and is rapidly displaced upon Brefeldin A exposition, indicating a loose association with Golgi membranes. A yeast two-hybrid screening revealed a specific interaction with the small GTPase ADP-ribosylation factor (ARF5) in its active, GTP-bound form. ARF5 and RAB6 bind to GORAB via the same internal Golgi-targeting RAB6 and ARF5 binding (IGRAB) domain. Two GORAB missense mutations identified in gerodermia osteodysplastica patients fall within this IGRAB domain. GORAB carrying the mutation p. Ala220Pro had a cytoplasmic distribution and failed to interact with both RAB6 and ARF5. In contrast, the p. Ser175Phe mutation displaced GORAB from the Golgi compartment to vesicular structures and selectively impaired ARF5 binding. Our findings indicate that the IGRAB domain is crucial for the Golgi localization of GORAB and that loss of this localization impairs its physiological function

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, β-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein–Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

Abstract Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, β-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates