BioID Performed on Golgi Enriched Fractions Identify C10orf76 as a GBF1 Binding Protein Essential for Golgi Maintenance and Secretion.

The Golgi-specific Brefeldin-A resistance factor 1 (GBF1) is the only large GEF that regulates Arf activation at the cis-Golgi and is actively recruited to membranes on an increase in Arf-GDP. Recent studies have revealed that GBF1 recruitment requires one or more heat-labile and protease-sensitive protein factor(s) (Quilty et al., 2018, J. Cell Science, 132). Proximity-dependent biotinylation (BioID) and mass spectrometry from enriched Golgi fractions identified GBF1 proximal proteins that may regulate its recruitment. Knockdown studies revealed C10orf76 to be involved in Golgi maintenance. We find that C10orf76 interacts with GBF1 and rapidly cycles on and off GBF1-positive Golgi structures. More importantly, its depletion causes Golgi fragmentation, alters GBF1 recruitment, and impairs secretion. Homologs were identified in most species, suggesting its presence in the last eukaryotic common ancestor.

The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor.

We previously proposed a novel mechanism by which the enzyme Golgi-specific Brefeldin A resistance factor 1 (GBF1) is recruited to the membranes of the cis-Golgi, based on in vivo experiments. Here, we extended our in vivo analysis on the production of regulatory Arf-GDP and observed that ArfGAP2 and ArfGAP3 do not play a role in GBF1 recruitment. We confirm that Arf-GDP localization is critical, as a TGN-localized Arf-GDP mutant protein fails to promote GBF1 recruitment. We also reported the establishment of an in vitro GBF1 recruitment assay that supports the regulation of GBF1 recruitment by Arf-GDP. This in vitro assay yielded further evidence for the requirement of a Golgi-localized protein because heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. Finally, combined in vivo and in vitro measurements indicated that the recruitment to Golgi membranes via a putative receptor requires only the HDS1 and HDS2 domains in the C-terminal half of GBF1.

Scyl1 scaffolds class II Arfs to specific subcomplexes of coatomer through the γ-COP appendage domain.

Coatomer (COPI)-coated vesicles mediate membrane trafficking in the early secretory pathway. There are at least three subclasses of COPI coats and two classes of Arf GTPases that couple COPI coat proteins to membranes. Whether mechanisms exist to link specific Arfs to specific COPI subcomplexes is unknown. We now demonstrate that Scy1-like protein 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, oligomerizes through centrally located HEAT repeats and uses a C-terminal RKXX-COO(-) motif to interact directly with the appendage domain of coatomer subunit γ-2 (also known as COPG2 or γ2-COP). Through a distinct site, Scyl1 interacts selectively with class II Arfs, notably Arf4, thus linking class II Arfs to γ2-bearing COPI subcomplexes. Therefore, Scyl1 functions as a scaffold for key components of COPI coats, and disruption of the scaffolding function of Scyl1 causes tubulation of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-Golgi, similar to that observed following the loss of Arf and Arf-guanine-nucleotide-exchange factor (GEF) function. Our data reveal that Scyl1 is a key organizer of a subset of the COPI machinery.

Arf activation at the Golgi is modulated by feed-forward stimulation of the exchange factor GBF1.

ADP-ribosylation factors (Arfs) play central roles in the regulation of vesicular trafficking through the Golgi. Arfs are activated at the Golgi membrane by guanine-nucleotide-exchange factors (GEFs) that are recruited from cytosol. Here, we describe a novel mechanism for the regulation of recruitment and activity of the ArfGEF Golgi-specific BFA resistance factor 1 (GBF1). Conditions that alter the cellular Arf-GDP:Arf-GTP ratio result in GBF1 recruitment. This recruitment of GBF1 occurs selectively on cis-Golgi membranes in direct response to increased Arf-GDP. GBF1 recruitment requires Arf-GDP myristoylation-dependent interactions suggesting regulation of a membrane-bound factor. Once recruited, GBF1 causes increased Arf-GTP production at the Golgi, consistent with a feed-forward self-limiting mechanism of Arf activation. This mechanism is proposed to maintain steady-state levels of Arf-GTP at the cis-Golgi during cycles of Arf-dependent trafficking events.

Chlamydia trachomatis co-opts GBF1 and CERT to acquire host sphingomyelin for distinct roles during intracellular development.

The strain designated Chlamydia trachomatis serovar that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis. [corrected]. The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM), a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA)-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM), for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic factory at or near the inclusion. We hypothesize that SM acquired by CERT-dependent transport of ceramide and subsequent conversion to SM is necessary for C. trachomatis replication whereas SM acquired by the GBF1-dependent pathway is essential for inclusion growth and stability. Our results reveal a novel mechanism by which an intracellular pathogen redirects SM biosynthesis to its replicative niche.

Evolution and diversity of the Golgi.

The Golgi is an ancient and fundamental eukaryotic organelle. Evolutionary cell biological studies have begun establishing the repertoire, processes, and level of complexity of membrane-trafficking machinery present in early eukaryotic cells. This article serves as a review of the literature on the topic of Golgi evolution and diversity and reports a novel comparative genomic survey addressing Golgi machinery in the widest taxonomic diversity of eukaryotes sampled to date. Finally, the article is meant to serve as a primer on the rationale and design of evolutionary cell biological studies, hopefully encouraging readers to consider this approach as an addition to their cell biological toolbox. It is clear that the major machinery involved in vesicle trafficking to and from the Golgi was already in place by the time of the divergence of the major eukaryotic lineages, nearly 2 billion years ago. Much of this complexity was likely generated by an evolutionary process involving gene duplication and coevolution of specificity encoding membrane-trafficking proteins. There have also been clear cases of loss of Golgi machinery in some lineages as well as innovation of novel machinery. The Golgi is a wonderfully complex and diverse organelle and its continued exploration promises insight into the evolutionary history of the eukaryotic cell.

Arf3 is activated uniquely at the trans-Golgi network by brefeldin A-inhibited guanine nucleotide exchange factors.

It is widely assumed that class I and II Arfs function interchangeably throughout the Golgi complex. However, we report here that in vivo, Arf3 displays several unexpected properties. Unlike other Golgi-localized Arfs, Arf3 associates selectively with membranes of the trans-Golgi network (TGN) in a manner that is both temperature-sensitive and uniquely dependent on guanine nucleotide exchange factors of the BIGs family. For example, BIGs knockdown redistributed Arf3 but not Arf1 from Golgi membranes. Furthermore, shifting temperature to 20 degrees C, a temperature known to block cargo in the TGN, selectively redistributed Arf3 from Golgi membranes. Arf3 redistribution occurred slowly, suggesting it resulted from a change in membrane composition. Arf3 knockdown and overexpression experiments suggest that redistribution is not responsible for the 20 degrees C block. To investigate in more detail the mechanism for Arf3 recruitment and temperature-dependent release, we characterized several mutant forms of Arf3. This analysis demonstrated that those properties are readily separated and depend on pairs of residues present at opposite ends of the protein. Furthermore, phylogenetic analysis established that all four critical residues were absolutely conserved and unique to Arf3. These results suggest that Arf3 plays a unique function at the TGN that likely involves recruitment by a specific receptor.

ADP-ribosylation factor 1 controls the activation of the phosphatidylinositol 3-kinase pathway to regulate epidermal growth factor-dependent growth and migration of breast cancer cells.

Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110alpha) to the plasma membrane, and the association of the regulatory subunit (p85alpha) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression.

Characterization of class I and II ADP-ribosylation factors (Arfs) in live cells: GDP-bound class II Arfs associate with the ER-Golgi intermediate compartment independently of GBF1.

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2-5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addition, loss of Arf x GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf x GTP, rather than the formation of an Arf x GDP x BFA x GBF1 complex.

Distinct functions for Arf guanine nucleotide exchange factors at the Golgi complex: GBF1 and BIGs are required for assembly and maintenance of the Golgi stack and trans-Golgi network, respectively.

We examined the relative function of the two classes of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factors that regulate recruitment of coat proteins on the Golgi complex. Complementary overexpression and RNA-based knockdown approaches established that GBF1 regulates COPI recruitment on cis-Golgi compartments, whereas BIGs appear specialized for adaptor proteins on the trans-Golgi. Knockdown of GBF1 and/or COPI did not prevent export of VSVGtsO45 from the endoplasmic reticulum (ER), but caused its accumulation into peripheral vesiculotubular clusters. In contrast, knockdown of BIG1 and BIG2 caused loss of clathrin adaptor proteins and redistribution of several TGN markers, but had no impact on COPI and several Golgi markers. Surprisingly, brefeldin A-inhibited guanine nucleotide exchange factors (BIGs) knockdown prevented neither traffic of VSVGtsO45 to the plasma membrane nor assembly of a polarized Golgi stack. Our observations indicate that COPII is the only coat required for sorting and export from the ER exit sites, whereas GBF1 but not BIGs, is required for COPI recruitment, Golgi subcompartmentalization, and cargo progression to the cell surface.

GBF1, a cis-Golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi protein traffic.

The formation and maturation of membrane carriers that transport cargo from the ER to the Golgi complex involves the sequential action of the coat protein complexes COPII and COPI. Recruitment of COPI to nascent carriers requires activation of ADP-ribosylation factors by a BrefeldinA-sensitive guanine nucleotide exchange factor. Using new antisera and a GFP-tagged protein, we demonstrate that the exchange factor GBF1 localized to both Golgi membranes and peripheral puncta, near but separate from ER exit sites. Live cell imaging revealed that GFP-GBF1 associates dynamically with both membranes through rapid exchange with a large cytosolic pool. Treatment with BrefeldinA dramatically altered this rapid exchange, causing accumulation of GBF1 on both Golgi and peripheral puncta before eventual redistribution to the ER in a microtubule-dependent manner. Measurement of diffusion coefficients and subcellular fractionation confirmed this shift in GBF1 from cytosolic to membrane bound. BrefeldinA-induced accumulation of GBF1 coincided with loss of COPI from peripheral puncta. Furthermore, recruitment of GBF1 to cargo-containing peripheral puncta coincided with recruitment of COPI, but not COPII. Strikingly, microinjection of anti-GBF1 antibodies specifically caused dissociation of COPI from membranes. These observations strongly suggest that GBF1 regulates COPI membrane recruitment in the early secretory pathway.

The Arf6 GEF GEP100/BRAG2 regulates cell adhesion by controlling endocytosis of beta1 integrins.

The small GTPase Arf6 has been shown to regulate the post-endocytic trafficking of a subset of membrane proteins, including beta1 integrins, and inhibition of Arf6 function impairs both cell adhesion and motility. The activity of Arf GTPases is regulated by a large family of guanine nucleotide exchange factors (GEFs). Arf-GEP100/BRAG2 is a GEF with reported specificity for Arf6 in vitro, but it is otherwise poorly characterized. Here we report that BRAG2 exists in two ubiquitously expressed isoforms, which we call BRAG2a and BRAG2b, both of which can activate Arf6 in vivo. Depletion of endogenous BRAG2 by siRNA leads to dramatic effects in the cell periphery; one such effect is an accumulation of beta1 integrin on the cell surface and a corresponding enhancement of cell attachment and spreading on fibronectin-coated substrates. In contrast, depletion of Arf6 leads to intracellular accumulation of beta1 integrin and reduced adhesion and spreading. These findings suggest that Arf6 regulates both endocytosis and recycling of beta1 integrins and that BRAG2 functions selectively to activate Arf6 during integrin internalization.

Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi.

Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Despite the implication of their neuronal isoforms in axonal regeneration, the function of their widely expressed isoforms is largely unknown. In this study, we examined the role of the ubiquitously expressed RTN3 in membrane trafficking. Ectopically expressed RTN3 exhibited heterogeneous patterns; filamentous, reticular, and granular distributions. The ER morphology changed accordingly. In cells where RTN3 displayed a filamentous/reticular distribution, protein transport between the ER and Golgi was blocked, and Golgi proteins were dispersed. In contrast, ERGIC-53, a marker for the ER-Golgi intermediate compartment, accumulated at the perinuclear region, and remained there even after cells were treated with agents that induce redistribution of Golgi proteins to the ER, indicating an inhibition of Golgi-to-ER transport of ERGIC-53. These results suggest that RTN3 plays a role in membrane trafficking in the early secretory pathway.

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf.

BACKGROUND: Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. RESULTS: Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7). CONCLUSION: Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

Exo1: a new chemical inhibitor of the exocytic pathway.

A phenotypic screen was used to search for drug-like molecules that can interfere with specific steps in membrane traffic. 2-(4-Fluorobenzoylamino)-benzoic acid methyl ester (Exo1), identified in this screen, induces a rapid collapse of the Golgi to the endoplasmic reticulum, thus acutely inhibiting the traffic emanating from the endoplasmic reticulum. Like Brefeldin A (BFA), Exo1 induces the rapid release of ADP-ribosylation factor (ARF) 1 from Golgi membranes but has less effect on the organization of the trans-Golgi network. Our data indicate that Exo1 acts by a different mechanism from BFA. Unlike BFA, Exo1 does not induce the ADP-ribosylation of CtBP/Bars50 and does not interfere with the activity of guanine nucleotide exchange factors specific for Golgi-based ARFs. Thus, Exo1 allows the fatty acid exchange activity of Bars50 to be distinguished from ARF1 activity in the control of Golgi tubulation.

Characterization of alternatively spliced and truncated forms of the Arf guanine nucleotide exchange factor GBF1 defines regions important for activity.

Analysis of multiple transcripts for the Arf-specific guanine nucleotide exchange factor GBF1 identified three positions displaying small in-frame deletions and insertions. Sequencing of genomic DNA for CHO GBF1 and analysis of the human gene established that those variations were consistent with alternate splicing events. RT-PCR analysis of CHO mRNA confirmed that these small in-frame deletions occurred at significant and similar frequencies in both WT and BFA resistant CHO cells. These splice variants behaved like GBF1 in biological assays based on the observation that GBF1 is cytotoxic at high levels but will confer resistance to BFA when moderately overexpressed. Comparison of variants with larger deletions defined regions of 75 (exons 5-7) and 412 (exons 31-39) amino acid residues that were required for cell killing but were dispensable for promoting BFA resistance.

Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic.

Activation of several ADP-ribosylation factors (ARFs) by guanine nucleotide exchange factors (GEFs) regulates recruitment of coat proteins (COPs) on the Golgi complex and is generally assumed to be the target of brefeldin A (BFA). The large ARF-GEFs Golgi-specific BFA resistance factor 1 (GBF1) and BFA-inhibited GEFs (BIGs) localize to this organelle but catalyze exchange preferentially on class II and class I ARFs, respectively. We now demonstrate using quantitative confocal microscopy that these GEFs show a very limited overlap with each other (15 and 23%). In contrast, GBF1 colocalizes with the cis-marker p115 (86%), whereas BIGs overlap extensively with TGN38 (83%). Consistent with these distributions, GBF1, but not BIG1, partially relocalized to peripheral sites after incubation at 15 degrees C. The new GBF1 structures represent peripheral vesicular tubular clusters (VTCs) because 88% of structures analyzed stained for both GBF1 and p115. Furthermore, as expected of VTCs, they rapidly reclustered to the Golgi complex in a microtubule-dependent manner upon warm-up. These observations suggest that GBF1 and BIGs activate distinct subclasses of ARFs in specific locations to regulate different types of reactions. In agreement with this possibility, COPI overlapped to a greater extent with GBF1 (64%) than BIG1 (31%), whereas clathrin showed limited overlap with BIG1, and virtually none with GBF1.