cis-Golgi Complex Research Articles

Golgi-localized Ring Finger Protein 121 is necessary for MYCN-driven neuroblastoma tumorigenesis

MYCN amplification predicts poor prognosis in childhood neuroblastoma. To identify MYCN oncogenic signal dependencies we performed N-ethyl-N-nitrosourea (ENU) mutagenesis on the germline of neuroblastoma-prone TH-MYCN transgenic mice to generate founders which had lost tumorigenesis. Sequencing of the mutant mouse genomes identified the Ring Finger Protein 121 (RNF121WT) gene mutated to RNFM158R associated with heritable loss of tumorigenicity. While the RNF121WT protein localised predominantly to the cis-Golgi Complex, the RNF121M158R mutation in Helix 4 of its transmembrane domain caused reduced RNF121 protein stability and absent Golgi localisation. RNF121WT expression markedly increased during TH-MYCN tumorigenesis, whereas hemizygous RNF121WT gene deletion reduced TH-MYCN tumorigenicity. The RNF121WT-enhanced growth of MYCN-amplified neuroblastoma cells depended on RNF121WT transmembrane Helix 5. RNF121WT directly bound MYCN protein and enhanced its stability. High RNF121 mRNA expression associated with poor prognosis in human neuroblastoma tissues and another MYC-driven malignancy, laryngeal cancer. RNF121 is thus an essential oncogenic cofactor for MYCN and a target for drug development.

Loss of hepatic SMLR1 causes hepatosteatosis and protects against atherosclerosis due to decreased hepatic VLDL secretion.

The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co-expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine-rich protein 1 ( SMLR1 ), encoding for small leucine-rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR-associated protein 9 gene editing to silence murine Smlr1 in hepatocytes ( Smlr1 -LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis-Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet-induced hyperlipidemia and atherosclerosis but causes NASH. On a high-fat, high-cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1 -LKO mice versus controls. We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis-Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.

Soluble guanylyl cyclase regulates skeletal muscle fiber type plasticity, fatigue resistance and whole body insulin resistance

Pathogenic defects in NO-cGMP signaling drive skeletal muscle dysfunction in Duchenne muscular dystrophy (DMD). These defects arise from decreased nNOS and guanylyl cyclase (GC) activity and loss of spatial control of NO production. Therapeutics such as sildenafil that amplify NO-cGMP signaling reduce skeletal muscle dysfunction in DMD patients and mouse models of DMD making GC and cGMP attractive therapeutic targets. However, GC and cGMP functions in skeletal muscle are poorly defined hindering therapy development. To remove this barrier, we investigated the functions of GC in skeletal muscle. We report that α1β1 soluble GC (sGC) is the primary cGMP source in skeletal muscle. α1β1 sGC expression was greater in oxidative muscles suggesting a greater cGMP synthesis capacity and muscle-specific differences in sGC function. Interestingly, sGC activity exhibited partial nNOS dependence. Analyses of sGC subcellular localization revealed a pool of α1β1 at the cis-Golgi complex in muscle cells. α1β1 and α2β1 sGC localized to the microvasculature. Muscles lacking a functional α1 sGC subunit (α1β1 sGC deficient) exhibited reduced fatigue resistance and normal hypertrophic growth. Surprisingly, α1β1 sGC deficiency had no impact on mitochondrial content suggesting that mitochondrial density may not be as tightly regulated by cGMP as previously thought. α1β1 sGC deficiency had a modest effect on mitochondrial ATP synthesis. Also, loss of α1β1 sGC triggered a type IIA to type IIX fiber shift. Although this shift was unlikely to significantly enhance fatigability, it may impact insulin metabolism because type IIX fiber content positively correlates with insulin insensitivity. Indeed, α1β1 sGC null mice exhibited gender-specific defects in whole body insulin sensitivity consistent with reports of gender-specific effects of cGMP on metabolism but normal glucose tolerance indicating compensatory changes in glucose metabolism. In summary, these findings argue that NO signaling through sGC plays important roles in muscle fatigue and fiber type specification and suggest sGC as a target for mitigating skeletal muscle fatigue in DMD. These data also suggest that disruption of NO-cGMP signaling may contribute to the poorly understood metabolic defects in DMD patients. By showing that reductions in cGMP synthesis promote muscle fatigue, type IIX content and insulin resistance, these data suggest new insights as to how decreases in cGMP may contribute to the development of metabolic dysfunction and disease, particularly over time. Importantly, these results suggest sGC as a potential target for mitigating insulin resistance.

Advances on Golgi glycoprotein 73 and its association with diseases

Golgi glycoprotein 73(GP73) is a transmembrane glycoprotein residing in the cis-Golgi complex, which is strongly expressed in hepatocellular carcinoma (HCC) and secreted into the blood. It has been regarded as a promising serum tumor marker for the detection of HCC with higher sensitivity and specificity than AFP. GP73 is also significantly elevated in kidney cancer, prostate cancer, lung adenocarcinoma, esophageal cancer and seminomas; therefore, it would be helpful for the diagnosis of these diseases. However, the function of GP73 and the regulatory mechanism for its expression are unclear. In this article, the physical-chemical properties, the regulation of its expression, the relation with various cancers and the clinical applications of GP73 are reviewed.

Intracellular Sequestration of the NKG2D Ligand ULBP3 by Human Cytomegalovirus

Human CMV (HCMV) encodes multiple genes that control NK cell activation and cytotoxicity. Some of these HCMV-encoded gene products modulate NK cell activity as ligands expressed at the cell surface that engage inhibitory NK cell receptors, whereas others prevent the infected cell from upregulating ligands that bind to activating NK cell receptors. A major activating NKR is the homodimeric NKG2D receptor, which has eight distinct natural ligands in humans. It was shown that HCMV is able to prevent the surface expression of five of these ligands (MIC A/B and ULBP1, 2, and 6). In this article, we show that the HCMV gene product UL142 can prevent cell surface expression of ULBP3 during infection. We further show that UL142 interacts with ULBP3 and mediates its intracellular retention in a compartment that colocalizes with markers of the cis-Golgi complex. In doing so, UL142 prevents ULBP3 trafficking to the surface and protects transfected cells from NK-mediated cytotoxicity. This is the first description of a viral gene able to mediate downregulation of ULBP3.

Dependence of Phospholipase D1 Multi-monoubiquitination on Its Enzymatic Activity and Palmitoylation

Phospholipase D (PLD) is an important lipase in many cellular processes, including vesicular trafficking, cell survival, and cell migration. In the present study, we show that PLD1, but not PLD2, is posttranslationally modified by multi-monoubiquitination. Intriguingly, suppression of lipase activity either by mutation of the HKD motif (PLD1 H896R, K898R, or D903A) or the phosphatidylinositol 4,5-bisphosphate binding motif (PLD1 R691G,R695G) or through use of PLD-selective inhibitors impaired the ubiquitination of PLD1, although stimulation of lipase activity by phorbol 12-myristate 13-acetate did not enhance its ubiquitination. A palmitoylation-deficient mutant PLD1 allele, which exhibits altered patterns of vesicular trafficking, had significantly lower levels of monoubiquitination. In addition, the expression of ubiquitin-fused PLD1 induced aberrantly enlarged vesicles partially co-localized with the Golgi complex but not with early endosomes. The altered localization was reduced by the K898R mutation, suggesting a role of multi-monoubiquitination in PLD1 subcellular localization. Surprisingly, the degradation of PLD1, but not of PLD1 K898R or PLD2, was blocked by inhibitors of proteasomes but not by inhibitors of lysosomes or other proteases, suggesting a role of the ubiquitination in proteasomal degradation of PLD1. In summary, our studies show that PLD1, but not PLD2, is multi-monoubiquitinated. The ubiquitination modification might represent a novel regulatory mechanism in PLD1 functioning, particularly in the context of subcellular trafficking between different membrane compartments.

Solution Structure of Human ζ-COP: Direct Evidences for Structural Similarity between COP I and Clathrin-Adaptor Coats

COP-I-coated vesicles are protein and lipid carriers that mediate intra-Golgi transport and transport from the cis-Golgi complex to the endoplasmic reticulum in cells. The coatomer of the vesicles coat is comprised of seven subunits: α-COP, ɛ-COP, β′-COP, β-COP, γ-COP, δ-COP, and ζ-COP. Here we report the solution structure of a truncated form (residues 1–149; ζ-COP149) of human ζ-COP (total 177 residues). It is the first three-dimensional structure of a “core” subunit of the COP I F-subcomplex. The structure of ζ-COP149 mainly consists of a disordered N-terminal tail, a five-stranded antiparallel β-sheet, a two-stranded antiparallel β-sheet, and five α-helices. The global folding of ζ-COP149 is very similar to the crystal structures of AP1-σ1 and AP2-σ2, directly demonstrating the structural similarity between the “core” subunits of the COP I F-subcomplex and adaptor protein complexes. Through structural comparison and mutagenesis study, we have also demonstrated that the heterodimers of ζ-COP149 and γ-COP have packing interfaces and relative subunit orientations similar to those of AP2-σ2 and AP2-α heterodimers. These results provide direct evidence supporting the previous proposal that the COP I F-subcomplex and adaptor protein complexes have similar tertiary and quaternary structures.

1H, 13C, and 15N resonance assignments of human ζ-COP

The zeta-COP is one subunit of the COP I coatomer, which mediates the protein trafficking from the cis-Golgi complex to the endoplasmic reticulum and also functions in the intra-Golgi trafficking. The NMR assignments of the zeta-COP are essential for its solution structure determination.

The perinuclear space of pancreatic acinar cells and the synthetic pathway of zymogen in Scorpaena scrofa L.: Ultrastructural aspects

Electron microscopic examination of exocrine pancreatic tissues from the fish Scorpaena scrofa L., probably captured while replenishing the acinar cells, shows two main functional cell morphologies of the same cell type. One cell functional aspect contains numerous well-contrasted small vesicles, the zymogenic vesicles. The other functional morphology is mainly represented by a few cells containing large apical zymogen vesicles with many empty RER cisterns. In our observations, the zymogenic vesicles are always studded with ribosomes. The main cytological finding is to report that zymogenic vesicles can be extruded from the perinuclear space and it confirms the suspected, synthetic activity of this cell compartment. The pool of zymogenic vesicles, maintaining their coat of ribosomes, then fuses and transfers their content into the cis Golgi complex network. Finally, the zymogen vesicles are produced following the classical secretory pathway from the trans Golgi saccular network into the supranuclear, apical region of the acinar cells where the largest vesicles concentrate their content until secretion.

ER-to-Golgi transport: COP I and COP II function (Review).

COP I and COP II coat proteins direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells. These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles. COP II proteins are required for selective export of newly synthesized proteins from the endoplasmic reticulum (ER). COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER. Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex.

Hepatitis C Virus Structural Proteins Reside in the Endoplasmic Reticulum as Well as in the Intermediate Compartment/cis-Golgi Complex Region of Stably Transfected Cells

The intracellular localization of hepatitis C virus structural proteins was analyzed by confocal immunofluorescence microscopy, cell fractionation, and immunoelectron microscopy in stably transfected cells that do not overexpress the viral proteins. The results strongly suggest that at steady state the structural proteins reside not only in the endoplasmic reticulum but also in the intermediate compartment and cis-Golgi complex region. By analogy with other viral systems, this finding raises the possibility that the intermediate compartment and cis-Golgi complex play a role in the assembly and budding of hepatitis C virus.

Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum.

Endoplasmic reticulum (ER) proteins maintain their residency by static retention, dynamic retrieval, or a combination of the two. Tail-anchored proteins that contain a cytosolic domain associated with the lipid bilayer via a hydrophobic stretch close to the COOH terminus are sorted within the secretory pathway by largely unknown mechanisms. Here, we have investigated the mode of insertion in the bilayer and the intracellular trafficking of cytochrome b(5) (b[5]), taken as a model for ER-resident tail-anchored proteins. We first demonstrated that b(5) can acquire a transmembrane topology posttranslationally, and then used two tagged versions of b(5), N-glyc and O-glyc b(5), containing potential N- and O-glycosylation sites, respectively, at the COOH-terminal lumenal extremity, to discriminate between retention and retrieval mechanisms. Whereas the N-linked oligosaccharide provided no evidence for retrieval from a downstream compartment, a more stringent assay based on carbohydrate acquisition by O-glyc b(5) showed that b(5) gains access to enzymes catalyzing the first steps of O-glycosylation. These results suggest that b(5) slowly recycles between the ER and the cis-Golgi complex and that dynamic retrieval as well as retention are involved in sorting of tail-anchored proteins.

Molecular characterization of caveolin association with the Golgi complex: identification of a cis-Golgi targeting domain in the caveolin molecule.

Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32-amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway.

A Different Intracellular Distribution of a Single Reporter Protein Is Determined at Steady State by KKXXor KDEL Retrieval Signals

To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C., Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R., and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex. In the latter compartments, CD8-E19 colocalizes with beta-coat protein (COP) (COPI component) and shows the higher density of labeling. Conversely, about 90% of the total CD8-KDEL protein is localized in clusters on the endoplasmic reticulum, where significant co-localization with Sec-23p (COPII component) is observed, and unglycosylated and initially O-glycosylated forms apparently constitute a single pool. Altogether, these results suggest that KKXX and KDEL retrieval motifs have different topological effects on theirs own at steady state: the first results in a specific enrichment in the intermediate compartment and cis-Golgi complex, and the latter dictates residency in the endoplasmic reticulum.

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion.

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.

The synaptobrevin-related domains of Bos1p and Sec22p bind to the syntaxin-like region of Sed5p.

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) are cytoplasmically oriented membrane proteins that reside on vesicular carriers (v-SNARE) and target organelles (t-SNARE). The pairing of a stage-specific v-SNARE with its cognate t-SNARE may mediate the specificity of membrane traffic. In the yeast Saccharomyces cerevisiae transport between the endoplasmic reticulum and Golgi complex employs two v-SNAREs, Bos1p and Sec22p, each containing a domain that is related to the neuronal v-SNARE synaptobrevin. Sed5p, which is homologous to syntaxin, is the t-SNARE that functions at this stage of the secretory pathway. Here we report that regions of Bos1p and Sec22p, which are homologous to synaptobrevin, bind to the syntaxin-like domain of Sed5p. Furthermore, we demonstrate that efficient v-SNARE/t-SNARE interactions require the participation of both v-SNAREs, indicating that, unlike post-Golgi membrane traffic, the active form of the endoplasmic reticulum to Golgi v-SNARE is a heteromeric complex.

Retention and degradation of proteins containing an uncleaved glycosylphosphatidylinositol signal.

Glycosylphosphatidylinositol (GPI) membrane anchor attachment is directed by a COOH-terminal signal that is proteolytically removed and replaced with a preformed GPI anchor in a coupled reaction. Failure to complete proteolytic cleavage and anchor addition results in the retention of an uncleaved precursor in a post-endoplasmic reticulum (ER) compartment. In this report, we address three issues: (i) the exact position of the transport block, (ii) the subsequent fate of the retained molecules, i.e. where are they degraded, and (iii) the mechanism whereby these proteins are selected for retention. Using decay accelerating factor (DAF), we provide evidence that failure to cleave the GPI signal totally prevents O-glycosylation, suggesting that the uncleaved polypeptides are not transported into the cis-Golgi complex. This implies that transport is blocked at the boundary between the ER-Golgi intermediate compartment and the Golgi stacks. The degradation of an intracellularly retained human growth hormone (hGH)-DAF fusion protein containing a nonfunctional GPI signal shows some features of ER degradation, i.e. the degradation is insensitive to leupeptin, chloroquine, and ammonium chloride, and is inhibited at 16 degrees C or after ATP depletion. However, morphological evidence points to a pathway resembling autophagy. To reconcile these observations, we suggest either that hGHDAF is degraded by two distinct pathways (ER degradation and autophagy) or that ER degradation takes place in an ER-associated vesicular compartment in a process resembling autophagy. Using as probes a soluble hGH receptor and an antibody recognizing only native hGH, we show that a significant fraction of the retained protein is correctly folded, ruling out general misfolding as the basis for retention. We also show that hGHDAF fusion proteins are present in high molecular weight, disulfide-linked aggregates in COS cells. We suggest a model for retention in which the uncleaved GPI signal drives the formation of large micelle-like aggregates that cannot be secreted.

GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.

The E1 glycoprotein of an avian coronavirus is targeted to the cis Golgi complex.

It was previously reported that the E1 protein of an avian coronavirus was targeted to the juxtanuclear region in COS cells expressing the protein from cloned cDNA, suggesting that the protein contains information for targeting to the Golgi complex. The first of three membrane-spanning domains was required for intracellular targeting, because a mutant E1 (delta m1,2) lacking this domain was delivered to the plasma membrane. We have used immunoelectron microscopy to localize the wild-type E1 protein within Golgi elements of COS cells and AtT-20 cells expressing these proteins from recombinant vaccinia vectors. By immunoperoxidase and immunogold labeling, the wild-type E1 protein was localized to one or two cisternae located on one side of the Golgi stack that could be identified as the cis side in AtT-20 cells. In contrast, the mutant E1 protein was detected in all cisternae across the stack as well as at the plasma membrane. When the E1 proteins were immunoprecipitated and subjected to digestion with endoglycosidase H, the majority of the wild-type E1 glycoprotein was endoglycosidase H sensitive, whereas the majority of the mutant E1 was processed to an endoglycosidase H-resistant, polylactosaminoglycan-containing form. The findings indicate that the wild-type E1 protein is specifically targeted to cis Golgi cisternae and are consistent with the assumption that the first membrane-spanning domain is required for targeting to the cis Golgi.

Influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate by skin fibroblasts.

The influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate from human skin fibroblasts was studied with the aid of a specific immunological procedure. Double-labeling experiments with [3H]leucine and [35S]sulfate indicated that monensin caused a dose-dependent parallel decrease of sulfate incorporation into total and of secretion of 3H-labeled proteodermatan sulfate. Compared with the untreated control, a greater proportion of incorporated [35S]sulfate than of incorporated [3H]leucine became secreted. Other monensin effects were a moderate intracellular accumulation of glycosaminoglycan-free core protein, a reduced chain length and a greatly reduced epimerization of D-glucuronic to L-iduronic acid residues. In contrast to the formation of N-acetylgalactosamine 4-sulfate residues 6-sulfation was not affected. Conversion of high-mannose-type oligosaccharides to complex-type N-glycans which normally occurred concomitantly with glycosaminoglycan biosynthesis was inhibited. Withdrawal of monensin made possible an additional sulfation of intracellularly accumulated proteodermatan sulfate. The newly formed sulfate esters did not cluster at the non-reducing ends of the glycosaminoglycan chains. Cells preexposed to monensin and labeled with [3H]glucosamine either in the absence or continuous presence of the drug incorporated similar amounts of 3H radioactivity into proteodermatan sulfate. The results suggest that epimerization of D-glucuronic acid residues and 4-sulfation occur predominantly in the trans cisternae of the Golgi apparatus whereas chain polymerisation and 6-sulfation take place predominantly in the cis Golgi complex.