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The endoplasmic reticulum protein Jagunal (JAGN1) was first identified as a requirement for Drosophila melanogaster oocyte development. Subsequent studies in human patients linked mutations in JAGN1 to severe congenital neutropenia, as well as a broad range of additional symptoms, suggesting that JAGN1 function is required in many tissues. Moreover, JAGN1 orthologs are found throughout animal and plant phylogeny, suggesting that JAGN1 supports fundamental cellular processes not restricted to egg development or neutrophil function. JAGN1 lacks sequence similarity or recognizable domains other than a coatomer protein complex I-binding motif, and its cellular function is currently unknown. JAGN1 shares a tetraspanning membrane topology with two families of known cargo transporters: the tetraspanins and the endoplasmic reticulum vesicle (Erv) proteins. Herein, we discuss the similarities between JAGN1, tetraspanins, and Ervs and, based on those, suggest a role for JAGN1 in facilitating the traffic of cell-restricted and ubiquitously expressed proteins at the endoplasmic reticulum-Golgi interface. [ABSTRACT FROM AUTHOR]

The ADP-ribosylation factor (ARF) superfamily of regulatory GTPases, including both the ARF and ARF-like (ARL) proteins, control a multitude of cellular functions, including aspects of vesicular traffic, lipid metabolism, mitochondrial architecture, the assembly and dynamics of the microtubule and actin cytoskeletons, and other pathways in cell biology. Considering their general utility, it is perhaps not surprising that increasingly ARF/ARLs have been found in connection to primary cilia. Here, we critically evaluate the current knowledge of the roles four ARF/ARLs (ARF4, ARL3, ARL6, ARL13B) play in cilia and highlight key missing information that would help move our understanding forward. Importantly, these GTPases are themselves regulated by guanine nucleotide exchange factors (GEFs) that activate them and by GTPase-activating proteins (GAPs) that act as both effectors and terminators of signaling. We believe that the identification of the GEFs and GAPs and better models of the actions of these GTPases and their regulators will provide a much deeper understanding and appreciation of the mechanisms that underly ciliary functions and the causes of a number of human ciliopathies. [ABSTRACT FROM AUTHOR]

Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by Golgi brefeldin A-resistant factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) with a highly conserved catalytic Sec7 domain (Sec7d). In addition to the Sec7d, GBF1 contains other conserved domains whose functions remain unclear. Here, we focus on HDS2 (homology downstream of Sec7d 2) domain because the L1246R substitution within the HDS2 α-helix 5 of the zebrafish GBF1 ortholog causes vascular hemorrhaging and embryonic lethality (13). To dissect the structure/function relationships within HDS2, we generated six variants, in which the most conserved residues within α-helices 1, 2, 4, and 6 were mutated to alanines. Each HDS2 mutant was assessed in a cell-based "replacement" assay for its ability to support cellular functions normally supported by GBF1, such as maintaining Golgi homeostasis, facilitating COPI recruitment, supporting secretion, and sustaining cellular viability. We show that cells treated with the pharmacological GBF1 inhibitor brefeldin A (BFA) and expressing a BFA-resistant GBF1 variant with alanine substitutions of RDR1168 or LF1266 are compromised in Golgi homeostasis, impaired in ARF activation, unable to sustain secretion, and defective in maintaining cellular viability. To gain insight into the molecular mechanism of this dysfunction, we assessed the ability of each GBF1 mutant to target to Golgi membranes and found that mutations in RDR1168 and LF1266 significantly decrease targeting efficiency. Thus, these residues within α-helix 2 and α-helix 6 of the HDS2 domain in GBF1 are novel regulatory determinants that support GBF1 cellular function by impacting the Golgi-specific membrane association of GBF1. [ABSTRACT FROM AUTHOR]

Members of the large Sec7 domain- containing Arf guanine nucleotide exchange factor (GEF) family have been shown to dimerize through their NH2-terminal dimerization and cyclophilin binding (DCB) and homology upstream of Sec7 (HUS) domains. However, the importance of dimerization in GEF localization and function has not been assessed. We generated a GBF1 mutant (91/130) in which two residues required for oligomerization (K91 and E130 within the DCB domain) were replaced with A and assessed the effects of these mutations on GBF1 localization and cellular functions. We show that 91/130 is compromised in oligomerization but that it targets to the Golgi in a manner indistinguishable from wild-type GBF1 and that it rapidly exchanges between the cytosolic and membrane-bound pools. The 91/130 mutant appears active as it integrates within the functional network at the Golgi, supports Arf activation and COPI recruitment, and sustains Golgi homeostasis and cargo secretion when provided as a sole copy of functional GBF1 in cells. In addition, like wild-type GBF1, the 91/130 mutant supports poliovirus RNA replication, a process requiring GBF1 but believed to be independent of GBF1 catalytic activity. However, oligomerization appears to stabilize GBF1 in cells, and the 91/130 mutant is degraded faster than the wild-type GBF1. Our data support a model in which oligomerization is not a key regulator of GBF1 activity but impacts its function by regulating the cellular levels of GBF1. [ABSTRACT FROM AUTHOR]