p115-SNARE interactions: a dynamic cycle of p115 binding monomeric SNARE motifs and releasing assembled bundles.

Tethering factors regulate the targeting of membrane-enclosed vesicles under the control of Rab GTPases. p115, a golgin family tether, has been shown to participate in multiple stages of ER/Golgi transport. Despite extensive study, the mechanism of action of p115 is poorly understood. SNARE proteins make up the machinery for membrane fusion, and strong evidence shows that function of p115 is directly linked to its interaction with SNAREs. Using a gel filtration binding assay, we have demonstrated that in solution p115 stably interacts with ER/Golgi SNAREs rbet1 and sec22b, but not membrin and syntaxin 5. These binding preferences stemmed from selectivity of p115 for monomeric SNARE motifs as opposed to SNARE oligomers. Soluble monomeric rbet1 can compete off p115 from coat protein II (COPII) vesicles. Furthermore, excess p115 inhibits p115 function in trafficking. We conclude that monomeric SNAREs are a major binding site for p115 on COPII vesicles, and that p115 dissociates from its SNARE partners upon SNAREpin assembly. Our results suggest a model in which p115 forms a mixed p115/SNARE helix bundle with a monomeric SNARE, facilitates the binding activity and/or concentration of the SNARE at prefusion sites and is subsequently ejected as SNARE complex formation and fusion proceed.

Identification of a functional domain within the p115 tethering factor that is required for Golgi ribbon assembly and membrane trafficking.

The tethering factor p115 (known as Uso1p in yeast) has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that depletion of p115 by using RNA interference (RNAi) in C. elegans causes accumulation of the 170 kD soluble yolk protein (YP170) in the body cavity and retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes. Structure-function analyses of p115 have identified two homology regions (H1 and H2) within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a new C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants that lack the fourth CC domain (CC4) act in a dominant-negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi of p115 and the subsequent transfection with p115 deletion mutants, we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115. p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function, and suggest that both the CC1 and the CC4 SNARE-binding motifs participate in p115-mediated membrane tethering.

On and off membrane dynamics of the endoplasmic reticulum-golgi tethering factor p115 in vivo.

The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t1/2 approximately 20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t1/2 approximately 13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP. p115 mutant missing the GM130/giantin binding site exhibits analogous fluorescence recovery after photobleaching (FRAP) (t1/2 approximately 13 s), suggesting that GM130 and giantin are not major determinants of p115 membrane dynamics. In contrast, p115-GFP exchanges more rapidly (t1/2 approximately 8 s) in cells expressing the inactive Rab1/N121I mutant, indicating that p115 cycling is influenced by Rab1. p115-GFP dynamics is also influenced by the assembly status of SNAREs. In cells expressing an ATPase-deficient NSF/E329Q mutant that inhibits SNARE complex disassembly, the cycling kinetics of p115-GFP are significantly slower (t1/2 approximately 21 s). In contrast, in cells incubated at reduced temperature (10 degrees C) that inhibits vesicular traffic, the cycling kinetics of p115-GFP are faster (t1/2 approximately 7 s). These data suggest that p115-binding sites on the membrane are provided by unassembled SNAREs. In agreement, biochemical studies show increased p115 recruitment to membranes in the presence of NSF and alpha-SNAP. Our data support a model in which recruitment of tethers is directly regulated by the assembly status of SNAREs.

Cytomegalovirus promotes intestinal macrophage-mediated mucosal inflammation through induction of Smad7.

Intestinal macrophages in healthy human mucosa are profoundly down-regulated for inflammatory responses (inflammation anergy) due to stromal TGF-ß inactivation of NF-κB. Paradoxically, in cytomegalovirus (CMV) intestinal inflammatory disease, one of the most common manifestations of opportunistic CMV infection, intestinal macrophages mediate severe mucosal inflammation. Here we investigated the mechanism whereby CMV infection promotes macrophage-mediated mucosal inflammation. CMV infected primary intestinal macrophages but did not replicate in the cells or reverse established inflammation anergy. However, CMV infection of precursor blood monocytes, the source of human intestinal macrophages in adults, prevented stromal TGF-ß-induced differentiation of monocytes into inflammation anergic macrophages. Mechanistically, CMV up-regulated monocyte expression of the TGF-ß antagonist Smad7, blocking the ability of stromal TGF-ß to inactivate NF-κB, thereby enabling MyD88 and NF-κB-dependent cytokine production. Smad7 expression also was markedly elevated in mucosal tissue from subjects with CMV colitis and declined after antiviral ganciclovir therapy. Confirming these findings, transfection of Smad7 antisense oligonucleotide into CMV-infected monocytes restored monocyte susceptibility to stromal TGF-ß-induced inflammation anergy. Thus, CMV-infected monocytes that recruit to the mucosa, not resident macrophages, are the source of inflammatory macrophages in CMV mucosal disease and implicate Smad7 as a key regulator of, and potential therapeutic target for, CMV mucosal disease.

Phosphorylation of Golgi Peripheral Membrane Protein Grasp65 Is an Integral Step in the Formation of the Human Cytomegalovirus Cytoplasmic Assembly Compartment.

Human cytomegalovirus (HCMV) is the largest member of the Herpesviridae and represents a significant cause of disease. During virus replication, HCMV alters cellular functions to facilitate its replication, including significant reorganization of the secretory and endocytic pathways of the infected cell. A defining morphologic change of the infected cell is the formation of a membranous structure in the cytoplasm that is designated the virion assembly compartment (AC), which consists of virion structural proteins surrounded by cellular membranes. The loss of normal Golgi compartment morphology and its relocalization from a juxtanuclear ribbonlike structure to a series of concentric rings on the periphery of the AC represents a readily recognized reorganization of cellular membranes in the HCMV-infected cell. Although trafficking of viral proteins to this compartment is required for the assembly of infectious virions, the functional significance of the reorganization of intracellular membranes like the Golgi membranes into the AC in the assembly of infectious virus remains understudied. In this study, we determined that Golgi membrane ribbon fragmentation increased during the early cytoplasmic phase of virion assembly and that Golgi membrane fragmentation in infected cells was dependent on the phosphorylation of an integral cis-Golgi protein, Grasp65. Inhibition of Golgi membrane fragmentation and of its reorganization into the AC resulted in decreased production of infectious particles and alteration of the incorporation of an essential protein into the envelope of the mature virion. These results demonstrated the complexity of the virus-host cell interactions required for efficient assembly of this large DNA virus. IMPORTANCE: The human cytomegalovirus (HCMV)-induced reorganization of intracellular membranes that is required for the formation of the viral assembly compartment (AC) has been an area of study over the last 20 years. The significance of this virus-induced structure has been evinced by the results of several studies which showed that relocalization of viral proteins to the AC was required for efficient assembly of infectious virus. In this study, we have identified a mechanism for the fragmentation of the Golgi ribbon in the infected cell en route to AC morphogenesis. Identification of this fundamental process during HCMV replication allowed us to propose that the functional role of Golgi membrane reorganization during HCMV infection was the concentration of viral structural proteins and subviral structures into a single intracellular compartment in order to facilitate efficient protein-protein interactions and the virion protein trafficking required for the assembly of this large and structurally complex virus.

Cytomegalovirus microRNAs.

The discovery that animals, plants and DNA viruses encode microRNAs (miRNAs) has transformed our understanding of the regulation of gene expression. miRNAs are ubiquitous small non-coding RNAs that regulate gene expression post-transcriptionally, generally by binding to sites within the 3' untranslated regions (UTR) of messenger RNA (mRNA) transcripts. To date, over 250 viral miRNAs have been identified primarily in members of the herpesvirus family. These viral miRNAs target both viral and cellular genes in order to regulate viral replication, the establishment and maintenance of viral latency, cell survival, and innate and adaptive immunity. This review will focus on our current knowledge of the targets and functions of human cytomegalovirus (HCMV) miRNAs and their functional equivalents in other herpesviruses.

Cytomegalovirus miRNAs target secretory pathway genes to facilitate formation of the virion assembly compartment and reduce cytokine secretion.

Herpesviruses, including human cytomegalovirus (HCMV), encode multiple microRNAs (miRNA) whose targets are just being uncovered. Moreover, miRNA function during the virus life cycle is relatively unknown. We find that HCMV miRs UL112-1, US5-1, and US5-2 target multiple components of the host secretory pathway, including VAMP3, RAB5C, RAB11A, SNAP23, and CDC42. A HCMV miR UL112-1, US5-1, and US5-2 triple mutant displayed aberrant morphogenesis of the virion assembly compartment (VAC), increased secretion of noninfectious particles, and increased IL-6 release from infected cells. Ectopic expression of miRs UL112-1, US5-1, and US5-2 or siRNAs directed against RAB5C, RAB11A, SNAP23, and CDC42 caused the loss of Golgi stacks with reorganization into structures that resemble the VAC and a decrease in cytokine release. These observations indicate that multiple HCMV miRNAs coordinately regulate reorganization of the secretory pathway to control cytokine secretion and facilitate formation of the VAC for efficient infectious virus production.

Tethering factor P115: a new model for tether-SNARE interactions.

The membrane tethering factor p115 has been shown to have important functions in ER to Golgi traffic and Golgi biogenesis. The multidomain structure of p115 allows for interactions with a diverse array of proteins that govern cargo movement at the ER-Golgi interface. Within its C-terminal region p115 contains four coiled-coil domains (CC1-CC4). Of the four coiled-coils, only CC1 has been shown to be required for p115 function, presumably by its ability to bind numerous SNARE proteins as well as the small GTPase Rab1. Recently, we showed that CC4 also interacts with SNARE proteins and that CC4 is required for p115 function in Golgi homeostasis and the trafficking of transmembrane but not soluble cargo. Here, we propose a novel model wherein p115 facilitates membrane tethering and fusion by simultaneously engaging its CC1 and CC4 domains with distinct SNARE proteins to promote formation of SNARE complexes.

Depletion of beta-COP reveals a role for COP-I in compartmentalization of secretory compartments and in biosynthetic transport of caveolin-1.

We have utilized small interfering RNA (siRNA)-mediated depletion of the beta-COP subunit of COP-I to explore COP-I function in organellar compartmentalization and protein traffic. Reduction in beta-COP levels causes the colocalization of markers for the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), Golgi, trans-Golgi network (TGN), and recycling endosomes in large, globular compartments. The lack of spatial differentiation of these compartments is not due to a general collapse of all cellular organelles since markers for the early endosomes and lysosomes do not redistribute to the common structures. Anterograde trafficking of the transmembrane cargo vesicular stomatitis virus membrane glycoprotein and of a subset of soluble cargoes is arrested within the common globular compartments. Similarly, recycling traffic of transferrin through the common compartment is perturbed. Furthermore, the trafficking of caveolin-1 (Cav1), a structural protein of caveolae, is arrested within the globular structures. Importantly, Cav1 coprecipitates with the gamma-subunit of COP-I, suggesting that Cav1 is a COP-I cargo. Our findings suggest that COP-I is required for the compartmentalization of the ERGIC, Golgi, TGN, and recycling endosomes and that COP-I plays a novel role in the biosynthetic transport of Cav1.

Dissecting the role of the ARF guanine nucleotide exchange factor GBF1 in Golgi biogenesis and protein trafficking.

COPI recruitment to membranes appears to be essential for the biogenesis of the Golgi and for secretory trafficking. Preventing COPI recruitment by expressing inactive forms of the ADP-ribosylation factor (ARF) or the ARF-activating guanine nucleotide exchange factor GBF1, or by treating cells with brefeldin A (BFA), causes the collapse of the Golgi into the endoplasmic reticulum (ER) and arrests trafficking of soluble and transmembrane proteins at the ER. Here, we assess COPI function in Golgi biogenesis and protein trafficking by preventing COPI recruitment to membranes by removing GBF1. We report that siRNA-mediated depletion of GBF1 causes COPI dispersal but does not lead to collapse of the Golgi. Instead, it causes extensive tubulation of the cis-Golgi. The Golgi-derived tubules target to peripheral ER-Golgi intermediate compartment (ERGIC) sites and create dynamic continuities between the ERGIC and the cis-Golgi compartment. COPI dispersal in GBF1-depleted cells causes dramatic inhibition of the trafficking of transmembrane proteins. Unexpectedly, soluble proteins continue to be secreted from GBF1-depleted cells. Our findings suggest that a secretory pathway capable of trafficking soluble proteins can be maintained in cells in which COPI recruitment is compromised by GBF1 depletion. However, the trafficking of transmembrane proteins through the existing pathway requires GBF1-mediated ARF activation and COPI recruitment.

Activation of ADP-ribosylation factor regulates biogenesis of the ATP7A-containing trans-Golgi network compartment and its Cu-induced trafficking.

ATP7A (MNK) regulates copper homeostasis by translocating from a compartment localized within the trans-Golgi network to the plasma membrane (PM) in response to increased copper load. The mechanisms that regulate the biogenesis of the MNK compartment and the trafficking of MNK are unclear. Here we show that the architecture of the MNK compartment is linked to the structure of the Golgi ribbon. Depletion of p115 tethering factor, which causes fragmentation of the Golgi ribbon, also disrupts the MNK compartment. In p115-depleted cells, MNK localizes to punctate structures that pattern on Golgi ministacks dispersed throughout the cell. Despite altered localization MNK trafficking still occurs, and MNK relocates from and returns to the fragmented compartment in response to copper. We further show that the biogenesis of the MNK compartment requires activation of ADP-ribosylation factor (Arf)1 GTPase, shown previously to facilitate the biogenesis of the Golgi ribbon. Activation of cellular Arf1 is prevented by 1) expressing an inactive "empty" form of Arf (Arf1/N126I), 2) expressing an inactive form of GBF1 (GBF1/E794K), guanine nucleotide exchange factor for Arf1, or 3) treating cells with brefeldin A, an inhibitor of GBF1 that disrupts MNK into a diffuse pattern. Importantly, preventing Arf activation inhibits copper-responsive trafficking of MNK to the PM. Our findings support a model in which active Arf is essential for the generation of the MNK compartment and for copper-responsive trafficking of MNK from there to the PM. Our findings provide an exciting foundation for identifying Arf1 effectors that facilitate the biogenesis of the MNK compartment and MNK traffic.

Mutations in COCH that result in non-syndromic autosomal dominant deafness (DFNA9) affect matrix deposition of cochlin.

The COCH gene mutated in autosomal dominant sensorineural deafness (DFNA9) encodes cochlin, a major constituent of the inner ear extracellular matrix. Sequence analysis of cochlin from DFNA9 patients identified five distinct single-amino-acid mutations within a conserved region (the LCCL domain) of cochlin. To define the molecular basis of DFNA9, we have generated myc-tagged wild-type and mutant cochlins and explored their behavior in transient transfection systems. Western blotting of cell lysates and culture media indicates that wild-type and mutant cochlins are synthesized and secreted in similar amounts. Immunofluorescent staining confirms that all are detected within the endoplasmic reticulum and the Golgi complex of transfected cells. Our findings suggest that COCH mutations are unlikely to cause abnormalities in secretion and suggest that extracellular events might cause DFNA9 pathology. In agreement, we show that wild-type cochlin accumulates in extracellular deposits that closely parallel the matrix component fibronectin, whereas mutant cochlins vary in the amount and pattern of extracellular material. Whereas some mutants exhibit an almost normal deposition pattern, some show complete lack of deposition. Our results suggest that DFNA9 results from gene products that fail to integrate correctly into the extracellular matrix. The partial or complete penetrance of integration defects suggests that DFNA9 pathology may be caused by multiple molecular mechanisms, including compromised ability of cochlin to self-assemble or to form appropriate complexes with other matrix components.