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1.
Martin Lowe 《The Journal of cell biology》2021,220(10)
In this issue of JCB, Welch et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202106115) show that GOLPH3 mediates the sorting of numerous Golgi proteins into recycling COPI transport vesicles. This explains how many resident proteins are retained at the Golgi and reveals a key role for GOLPH3 in maintaining Golgi homeostasis.The Golgi apparatus lies at the heart of the secretory pathway, where its major functions are the posttranslational modification of cargo proteins and lipids, particularly at the level of glycosylation, and the sorting of cargo to its correct onward destination. The Golgi is composed of stacked membrane compartments called cisternae, which contain numerous resident enzymes that act on the cargo as it passes through the organelle, from the entry or cis side to the exit or trans side. Each resident enzyme has its own distribution within the Golgi stack, resulting in the sequential modification of the secretory cargo as it moves through the Golgi.Various mechanisms exist to ensure that Golgi residents are retained within the Golgi despite the huge flux of protein and lipid through this organelle (1). Major players are COPI vesicles, which recycle Golgi residents from later to earlier cisternae, at the same time as the cisternae are thought to slowly migrate across the stack, as on a conveyor belt, progressively changing composition in a process referred to as cisternal maturation (2). Unlike the Golgi resident enzymes, which enter recycling vesicles, cargo is thought to remain within the maturing cisternae as it moves through the Golgi. Certain Golgi enzymes can bind directly to the COPI coat, explaining their inclusion in COPI vesicles (3), but for other enzymes and resident proteins, their retention mechanism is less obvious.Previous studies on the peripheral Golgi membrane protein GOLPH3 and its paralogue GOLPH3L (herein I will refer to both proteins as GOLPH3) indicated it can bind to certain Golgi enzymes and to the COPI coat, thereby acting as an adaptor to mediate sorting of these enzymes into COPI vesicles (4, 5). This was first shown for the yeast orthologue Vps74p (6, 7) and has also been demonstrated for the Drosophila version of the protein (8), consistent with a conserved function in Golgi enzyme retention. However, the extent to which GOLPH3 might participate in retention of different Golgi enzymes and other resident proteins, and its importance relative to other methods of protein retention in the Golgi, has remained unclear. Indeed, a recent study suggested that GOLPH3 selectively mediates the retention of enzymes involved in glycosphingolipid synthesis, consistent with a fairly selective role in retaining only a subset of resident Golgi enzymes (9). It should also be noted that GOLPH3 has been implicated in other functions, namely budding of exocytic vesicles from the Golgi, the DNA damage response, and mechanistic target of rapamycin signaling (10).In their current paper, Welch et al. used a combination of approaches to reassess the role of GOLPH3 at the Golgi (11). Using proteomics, they could identify numerous GOLPH3 binding partners, which included COPI, as expected, and a large number of other Golgi residents, including numerous Golgi enzymes and other membrane proteins. The ability of GOLPH3 to retain enzymes at the Golgi was confirmed using microscopy and an innovative flow cytometry–based assay to quantify surface versus Golgi abundance. The large number of possible interactors suggested that GOLPH3 could mediate the Golgi retention of many proteins. To further assess this possibility, the authors took advantage of previous observations showing that Golgi enzymes may be misrouted to the lysosome and degraded upon their failure to be retained in the Golgi (6, 7, 9). Using mass spectrometry, they could show that numerous Golgi resident proteins were depleted in GOLPH3 knockout cells, many of which were also found in the GOLPH3 interactome. This included many enzymes involved in glycosylation, consistent with GOLPH3 playing an important role in maintaining Golgi-dependent glycosylation of proteins and lipids. This was supported by lectin analysis, which showed marked changes in a broad range of glycans in the GOLPH3 knockout cells.The large number of GOLPH3 clients raises the question as to how it can recognize so many proteins. Previous work has shown binding to the cytoplasmic tails of Golgi enzymes and an interaction motif has been described for Vps74p and more recently for GOLPH3 (6, 9). However, bioinformatics analysis of the many GOLPH3 clients combined with mutational analysis, as performed in the current study, revealed the lack of a consensus sequence for GOLPH3 binding, with the common feature being a strong net positive charge combined with short cytoplasmic tail length. This would result in a high positive charge proximal to the membrane, which likely allows interaction with an acidic patch on the surface of GOLPH3. This mode of binding could mediate selective retention of many Golgi residents, while allowing for the forward trafficking of cargo proteins that have longer, less charged, or folded cytoplasmic domains.GOLPH3 is an oncogene associated with many types of cancer (12). Several mechanisms have been proposed to account for the oncogenic properties of GOLPH3, but most compelling is that changes in glycosylation are responsible. It was recently shown that GOLPH3-dependent changes in glycosphingolipids affects cell growth by altering mitogenic signaling (9). Changes in glycosylation of surface receptors has also been reported, which can affect surface abundance and hence signaling (13). The new results from Welch et al. suggest that glycosylation of many proteins and lipids may be relevant in cancer and that potentially a broad range of downstream targets contribute to oncogenesis. Such targets could influence processes beyond signaling, including cell adhesion and migration, that are known to be sensitive to changes in the surface glycome and which have been reported in previous studies on GOLPH3 (12).The study by Welch et al. indicates a major role for GOLPH3 in Golgi protein retention (Fig. 1). Clearly though, other retention mechanisms exist, including direct binding to COPI, and transmembrane domain length is also important, where the short transmembrane domain of resident proteins favors partitioning into recycling COPI vesicles and Golgi cisternal membranes of a similar thickness (1). Additional COPI adaptors are also likely, with TM9SF2 recently identified as a likely candidate, being present in Golgi vesicles and able to bind certain Golgi enzymes (1). It is possible that different resident proteins use different adaptors, or that a combination of retention mechanisms act in conjunction for certain residents, providing robustness to the retention process. However, any redundancy would seem incomplete given the strong phenotype seen upon loss of GOLPH3. GOLPH3 is localized toward the trans side of the Golgi, so it is possible that other adaptors, such as TM9SF2 and possibly others, might act earlier in the Golgi, or that direct coat binding is more important within the early Golgi. Hence different residents may be more likely to use different retention mechanisms depending on their location in the Golgi. Because GOLPH3 acts late in the Golgi and can bind many clients, we may think of it as a gatekeeper to prevent loss of numerous Golgi residents from the organelle.Open in a separate windowFigure 1.GOLPH3 plays a major role in Golgi protein retention. Golgi resident proteins, including many glycosylation enzymes, depicted by lollipops, are sorted into recycling COPI vesicles to maintain retention in the Golgi in the face of onward cisternal maturation and secretory cargo transport. Different enzymes are depicted by different lollipop shapes and colors, with GOLPH3 clients indicated by horizontal ovals. Enzymes retained by other mechanisms are depicted by lollipops with circles (transmembrane domain length), squares or vertical ovals (binding to other COPI adaptors, indicated in turquoise and purple), or hexagons (direct binding to the COPI coat). GOLPH3, which is more abundant toward the trans side of the Golgi, has many clients.With regard to possible future studies, although we have a good idea of how GOLPH3 recognizes its clients, detailed structural analysis will prove informative in elucidating how it can bind so many proteins. Similarly, identification of additional adaptors linking Golgi residents to the COPI coat will be important to generate a more comprehensive view of Golgi protein retention. Finally, in the context of disease, further analysis of the glycoproteins and glycolipids whose levels are altered because of changes in GOLPH3 expression, of which there are likely to be many, should provide significant new insights into the mechanisms underlying GOLPH3-mediated tumorigenesis. 相似文献
2.
3.
Golgi phosphoprotein 2 (GOLPH2/GP73/GOLM1), a type-II Golgi transmembrane protein of unknown function, is up-regulated in
many cancers. Its Golgi luminal domain is potentially the major functional domain. The goal of this study is to identify the
proteins interacting with GOLPH2. Using secretory GOLPH2 (sGOLPH2, amino acid residues 55–401) as bait, secretory clusterin
(sCLU) was identified as one interacting candidate by yeast two-hybrid screening, and the coiled-coil domain of GOLPH2 was
found to be sufficient for interaction with sCLU. The interaction between GOLPH2 and sCLU was confirmed intracellularly and
extracellularly. The intracellular co-localization of GOLPH2 and sCLU in Golgi was also shown. These results can help in understanding
the biological and pathological significance of GOLPH2. 相似文献
4.
María J. Tenorio Breyan H. Ross Charlotte Luchsinger Andrés Rivera-Dictter Cecilia Arriagada Diego Acu?a Marcelo Aguilar Viviana Cavieres Patricia V. Burgos Pamela Ehrenfeld Gonzalo A. Mardones 《PloS one》2016,11(4)
Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes. 相似文献
5.
Xiaodong Guo Lu Xiong Lin Zou Ting Sun Jing Zhang Hanwei Li Ruiyun Peng Jingmin Zhao 《Diagnostic pathology》2012,7(1):1-7
Background
To investigate the expression of Golgi phosphoprotein-3 (GOLPH3) in prostate cancer and determine its prognostic value.Methods
Immunohistochemical staining for GOLPH3 was performed on tissue microarrays of 342 prostate patients. The correlation between GOLPH3 expression with its clinicopathologic factors was also analyzed in order to determine its prognostic significance.Results
GOLPH3 expression of normal prostate tissues, benign prostate hyperplasia, high-grade prostatic intraepithelial neoplasia, and hormone-dependent prostate cancer (HDPC) did not show any statistically significant difference. In contrast, statistically significant difference was reported in moderate/intense GOLPH3 expression in cases diagnosed with HDPC and castration resistant prostate cancer (CRPC) (P < 0.0005). Moderate /intense expression of GOLPH3 was associated with androgen independence (P?=?0.012), higher Gleason score (P?=?0.017), bone metastasis (P?=?0.024), higher baseline prostate-specific antigen (PSA) (P?=?0.038), and higher PSA nadir (P?=?0.032). A significantly negative correlation was found between moderate/intense GOLPH3 expression and disease-free survival (DFS) (HR?=?0.28, P?=?0.012) and overall survival (OS) (HR?=?0.42, P?=?0.027). Univariated analysis indicated that moderate/intense GOLPH3 expression created a significantly prognostic impact in patients with CRPC. On the other hand, multivariate analysis indicated that GOLPH3 was a significantly independent prognostic factor of DFS (P?=?0.027) in all prostate cancer patients.Conclusions
In this study, it was discovered that the overexpression of GOLPH3 is associated with the transition of prostate cancer from hormone sensitive phase to hormone refractory phase. GOLPH3 might be an important prognostic factor of DFS and OS in patients with prostate cancer. In totality, GOLPH3 could be used as a novel candidate in devising a more effective therapeutic strategy to tackle CRPC.Virtual slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1452541171722856. 相似文献6.
Gong Y Long Q Xie H Zhang T Peng T 《Biochemical and biophysical research communications》2012,421(4):713-720
Human Golgi phosphoprotein 2 gene (also known as GOLPH2, GP73 or GOLM1) encodes an epithelial-specific Golgi membrane protein which can be induced by virus infection. It is also overexpressed in a number of tumors and is currently considered as an early diagnosis marker for hepatocellular carcinoma. However, little is known about how GOLPH2 is dysregulated in these disease conditions and the functional implications of its overexpression. The aim of this study is to investigate human GOLPH2 regulation mechanisms. We cloned a 2599 bp promoter fragment of GOLPH2 and found it maintained epithelial specificity. By deletion analysis, a repressive region (-864 to -734 bp), a positive regulatory region (-734 to -421 bp) and a core promoter region (-421 to -79 bp) were identified. Sequence analysis revealed that GOLPH2 core promoter was devoid of canonical TATA element and classified as a TATA-less promoter. Adenoviral early region 1A (E1A) was able to activate GOLPH2 and the CtBP interaction domain of E1A was sufficient but not required for activation. A GC-box motif (-89 to -83 bp) in GOLPH2 core promoter region partly mediated E1A transactivation. These results delineated regulatory regions and functional element in GOLPH2 promoter, elucidated adenoviral E1A stimulation mechanisms and provided insight into GOLPH2 functions. 相似文献
7.
Michelle M. Ng Holly C. Dippold Matthew D. Buschman Christopher J. Noakes Seth J. Field 《Molecular biology of the cell》2013,24(6):796-808
GOLPH3 is a phosphatidylinositol-4-phosphate (PI4P) effector that plays an important role in maintaining Golgi architecture and anterograde trafficking. GOLPH3 does so through its ability to link trans-Golgi membranes to F-actin via its interaction with myosin 18A (MYO18A). GOLPH3 also is known to be an oncogene commonly amplified in human cancers. GOLPH3L is a GOLPH3 paralogue found in all vertebrate genomes, although previously it was largely uncharacterized. Here we demonstrate that although GOLPH3 is ubiquitously expressed in mammalian cells, GOLPH3L is present in only a subset of tissues and cell types, particularly secretory tissues. We show that, like GOLPH3, GOLPH3L binds to PI4P, localizes to the Golgi as a consequence of its PI4P binding, and is required for efficient anterograde trafficking. Surprisingly, however, we find that perturbations of GOLPH3L expression produce effects on Golgi morphology that are opposite to those of GOLPH3 and MYO18A. GOLPH3L differs critically from GOLPH3 in that it is largely unable to bind to MYO18A. Our data demonstrate that despite their similarities, unexpectedly, GOLPH3L antagonizes GOLPH3/MYO18A at the Golgi. 相似文献
8.
Golgi phosphoprotein 2 (GOLPH2) is a resident Golgi type-II membrane protein upregulated in liver disease. Given that GOLPH2 traffics through endosomes and can be secreted into the circulation, it is a promising serum marker for liver diseases. The structure of GOLPH2 and the functions of its different protein domains are not known. In the current study, we investigated the structural determinants for Golgi localization using a panel of GOLPH2 truncation mutants. The Golgi localization of GOLPH2 was not affected by the deletion of the C-terminal part of the protein. A truncated mutant containing the N-terminal portion (the cytoplasmic tail and transmembrane domain (TMD)) localized to the Golgi. Sequential deletion analysis of the N-terminal indicated that the TMD with a positively charged residue in the cytoplasmic N-terminal tail were sufficient to support Golgi localization. We also showed that both endogenous and secreted GOLPH2 exist as a disulfide-bonded dimer, and the coiled-coil domain was sufficient for dimerization. This structural knowledge is important for the understanding the pathogenic role of GOLPH2 in liver diseases, and the development of GOLPH2-based hepatocellular cancer diagnostic methods. 相似文献
9.
Study Design
To investigate the specific mechanisms by which Golgi phosphoprotein 3 (GOLPH3) affects the progression of gastric cancer and to explore its clinical significance.Methods
Immunohistochemical analysis was used to evaluate the correlations between GOLPH3, phosphorylated mTOR (p-mTOR), phosphorylated Akt (p-Akt), phosphorylated p70S6 (p-p70S6), phosphorylated 4E-BP1 (p-4E-BP1) and the clinicopathological features of gastric cancer. The mRNA expression levels of GOLPH3, mTOR, Akt, p70S6 and 4E-BP1 in gastric cancer, carcinoma-adjacent and paired normal tissue were analyzed using RT-PCR. Western blotting was used to determine the protein expression of GOLPH3, p-mTOR, p-Akt, p-p70S6 and p-4E-BP1 in tissues.Results
High expression protein levels of GOLPH3, p-AKT, p-mTOR, p70S6, p-4E-BP1 were positively associated with histological grade (p<0.05), depth of invasion (p<0.05), distant metastasis (p<0.05) and lymph node involvement (p<0.05). Compared with carcinoma-adjacent and paired normal tissues, the mRNA expression levels of GOLPH3, AKT, mTOR, p70S6 and 4EBP1 in gastric cancer tissues were significantly higher. The protein expression levels of GOLPH3, p-AKT, p-mTOR, p-p70S6 and p-4E-BP1 in gastric cancer tissues were also significantly higher than in carcinoma-adjacent and paired normal tissues. A strong positive correlation was observed between GOLPH3, p-mTOR, p-p70S6 and p-4EBP1 expression (r = 0.410, 0.303 and 0.276, respectively, p<0.05), but no significant correlation between the expression of GOLPH3 and p-Akt was observed.Conclusions
The GOPLH3 expression level is highly correlated with Akt/mTOR signaling in human gastric cancer samples. GOLPH3 combined with Akt/mTOR signaling activation may play an important role in the development, differentiation, invasion and metastasis of gastric cancer. 相似文献10.
Jun-Wei Song Jing Zhu Xing-Xuan Wu Ting Tu Jing-Qiang Huang Guan-Zi Chen Li-Yin Liang Chun-Hui Zhou XingZhi Xu Li-Yun Gong 《Cell death & disease》2021,12(11)
Cancer metastasis is the main cause of mortality associated with non-small-cell lung cancer (NSCLC), accounting for up to 70% of deaths among patients. The mechanisms underlying distal metastasis remain largely unknown. Golgi phosphoprotein 3 (GOLPH3) correlates negatively with overall survival in multiple tumors. In this study, we evaluated the function of GOLPH3 in NSCLC distal metastasis. GOLPH3 was expressed at high levels in samples from patients with NSCLC and was positively associated with clinicopathologic characteristics including clinical stage (P < 0.001), T (P = 0.001), N (P = 0.007), and M (P = 0.001) classification. Functionally, Transwell and wound-healing assays suggested that GOLPH3 overexpression enhances NSCLC cell migration and invasion abilities. Tumor-sphere formation and flow cytometry assays demonstrated that GOLPH3 overexpression enhances a stem cell-like phenotype of NSCLC cells. Metastasis models established by tail vein and intracardiac injection confirmed the pro-metastatic function of GOLPH3 in vivo. A subcutaneous tumor formation model confirmed that GOLPH3 overexpression increased the tumorigenicity of NSCLC cells. Mechanistically, gene set enrichment analysis revealed a positive association of GOLPH3 mRNA expression with WNT-activated gene signatures. Luciferase-reporter and nuclear extract assays showed that GOLPH3 overexpression enhances metastasis and tumorigenicity through activation of the WNT/β-catenin pathway. Immunoprecipitation-mass spectrometry and gene ontology analysis demonstrated that GOLPH3 interacts with cytoskeleton-associated protein 4 (CKAP4) in exosome-mediated distal metastasis. We found that GOLPH3 decreased the amount of plasma membrane-localized CKAP4 and increased the amount of exosome-localized CKAP4 to promote the formation of CKAP4-containing exosomes. Furthermore, we demonstrated that CKAP4 binds exosomal WNT3A to enhance its secretion. Therefore, the GOLPH3/CKAP4 axis plays a crucial role in promoting exosomal-WNT3A secretion to enhance and maintain the stem-like phenotype and metastasis in NSCLC, thus indicating the therapeutic potential of GOLPH3 in patients with NSCLC metastasis.Subject terms: Tumour biomarkers, Cell invasion 相似文献
11.
Lawrence G. Welch Sew-Yeu Peak-Chew Farida Begum Tim J. Stevens Sean Munro 《The Journal of cell biology》2021,220(10)
The fidelity of Golgi glycosylation is, in part, ensured by compartmentalization of enzymes within the stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterization of the clientele of GOLPH3 and its paralogue GOLPH3L is lacking. GOLPH3’s role is of particular interest as it is frequently amplified in several solid tumor types. Here, we apply two orthogonal proteomic methods to identify GOLPH3+3L clients and find that they act in diverse glycosylation pathways or have other roles in the Golgi. Binding studies, bioinformatics, and a Golgi retention assay show that GOLPH3+3L bind the cytoplasmic tails of their clients through membrane-proximal positively charged residues. Furthermore, deletion of GOLPH3+3L causes multiple defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi. 相似文献
12.
Stefano Sechi Gianni Colotti Giorgio Belloni Vincenzo Mattei Anna Frappaolo Grazia D. Raffa Margaret T. Fuller Maria Grazia Giansanti 《PLoS genetics》2014,10(5)
The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein has been described as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is also known as a potent oncogene, commonly amplified in several human tumors. However, the molecular pathways through which the oncoprotein GOLPH3 acts in malignant transformation are largely unknown. GOLPH3 has never been involved in cytokinesis. Here, we characterize the Drosophila melanogaster homologue of human GOLPH3 during cell division. We show that GOLPH3 accumulates at the cleavage furrow and is required for successful cytokinesis in Drosophila spermatocytes and larval neuroblasts. In premeiotic spermatocytes GOLPH3 protein is required for maintaining the organization of Golgi stacks. In dividing spermatocytes GOLPH3 is essential for both contractile ring and central spindle formation during cytokinesis. Wild type function of GOLPH3 enables maintenance of centralspindlin and Rho1 at cell equator and stabilization of Myosin II and Septin rings. We demonstrate that the molecular mechanism underlying GOLPH3 function in cytokinesis is strictly dependent on the ability of this protein to interact with PI(4)P. Mutations that abolish PI(4)P binding impair recruitment of GOLPH3 to both the Golgi and the cleavage furrow. Moreover telophase cells from mutants with defective GOLPH3-PI(4)P interaction fail to accumulate PI(4)P-and Rab11-associated secretory organelles at the cleavage site. Finally, we show that GOLPH3 protein interacts with components of both cytokinesis and membrane trafficking machineries in Drosophila cells. Based on these results we propose that GOLPH3 acts as a key molecule to coordinate phosphoinositide signaling with actomyosin dynamics and vesicle trafficking during cytokinesis. Because cytokinesis failures have been associated with premalignant disease and cancer, our studies suggest novel insight into molecular circuits involving the oncogene GOLPH3 in cytokinesis. 相似文献
13.
Tomoya Isaji Sanghun Im Wei Gu Yuqin Wang Qinglei Hang Jishun Lu Tomohiko Fukuda Noritaka Hashii Daisuke Takakura Nana Kawasaki Hiroyuki Miyoshi Jianguo Gu 《The Journal of biological chemistry》2014,289(30):20694-20705
Recently, the Golgi phosphoprotein 3 (GOLPH3) and its yeast homolog Vps74p have been characterized as essential for the Golgi localization of glycosyltransferase in yeast. GOLPH3 has been identified as a new oncogene that is commonly amplified in human cancers to modulate mammalian target of rapamycin signaling. However, the molecular mechanisms of the carcinogenic signaling pathway remain largely unclear. To investigate whether the expression of GOLPH3 was involved in the glycosylation processes in mammalian cells, and whether it affected cell behavior, we performed a loss-of-function study. Cell migration was suppressed in GOLPH3 knockdown (KD) cells, and the suppression was restored by a re-introduction of the GOLPH3 gene. HPLC and LC/MS analysis showed that the sialylation of N-glycans was specifically decreased in KD cells. The specific interaction between sialyltransferases and GOLPH3 was important for the sialylation. Furthermore, overexpression of α2,6-sialyltransferase-I rescued cell migration and cellular signaling, both of which were blocked in GOLPH3 knockdown cells. These results are the first direct demonstration of the role of GOLPH3 in N-glycosylation to regulate cell biological functions. 相似文献
14.
Mohamed F. Ali Vishwanath B. Chachadi Armen Petrosyan Pi-Wan Cheng 《The Journal of biological chemistry》2012,287(47):39564-39577
Core 2 N-acetylglucosaminyltransferase 1 (C2GnT1) is a key enzyme participating in the synthesis of core 2-associated sialyl Lewis x (C2-O-sLex), a ligand involved in selectin-mediated leukocyte trafficking and cancer metastasis. To accomplish that, C2GnT1 needs to be localized to the Golgi and this step requires interaction of its cytoplasmic tail (CT) with a protein that has not been identified. Employing C2GnT1 CT as the bait to perform a yeast two-hybrid screen, we have identified Golgi phosphoprotein 3 (GOLPH3) as a principal candidate protein that interacts with C2GnT1 and demonstrated that C2GnT1 binds to GOLPH3 via the LLRRR9 sequence in the CT. Confocal fluorescence microscopic analysis shows substantial Golgi co-localization of C2GnT1 and GOLPH3. Upon GOLPH3 knockdown, C2GnT1 is found mainly in the endoplasmic reticulum and decorated with complex-type N-glycans, indicating that the enzyme has been transported to the Golgi but is not retained. Also, we have found that a recombinant protein consisting of C2GnT1 CT1–16-Leu17–32-Gly33–42-GFP is localized to the Golgi although the same construct with mutated CT (AAAAA9) is not. The data demonstrate that the C2GnT1 CT is necessary and sufficient for Golgi localization of C2GnT1. Furthermore, GOLPH3 knockdown results in reduced synthesis of C2-O-sLex associated with P-selectin glycoprotein ligand-1, reduced cell tethering to and rolling on immobilized P- or E-selectin, and compromised E-selectin-induced activation of spleen tyrosine kinase and cell adhesion to intercellular adhesion molecule-1 under dynamic flow. Our results reveal that GOLPH3 can regulate cell-cell interaction by controlling Golgi retention of C2GnT1. 相似文献
15.
Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process. 相似文献
16.
Elias S. P. Eckert Ingeborg Reckmann Andrea Hellwig Simone R?hling Assou El-Battari Felix T. Wieland Vincent Popoff 《The Journal of biological chemistry》2014,289(45):31319-31329
Newly synthesized membrane and secreted proteins undergo a series of posttranslational modifications in the Golgi apparatus, including attachment of carbohydrate moieties. The final structure of so-formed glycans is determined by the order of execution of the different glycosylation steps, which seems intimately related to the spatial distribution of glycosyltransferases and glycosyl hydrolases within the Golgi apparatus. How cells achieve an accurate localization of these enzymes is not completely understood but might involve dynamic processes such as coatomer-coated (COPI) vesicle-mediated trafficking. In yeast, this transport is likely to be regulated by vacuolar protein sorting 74 (Vps74p), a peripheral Golgi protein able to interact with COPI coat as well as with a binding motif present in the cytosolic tails of some mannosyltransferases. Recently, Golgi phosphoprotein 3 (GOLPH3), the mammalian homolog of Vps74, has been shown to control the Golgi localization of core 2 N-acetylglucosamine-transferase 1. Here, we highlight a role of GOLPH3 in the spatial localization of α-2,6-sialyltransferase 1. We show, for the first time, that GOLPH3 supports incorporation of both core 2 N-acetylglucosamine-transferase 1 and α-2,6-sialyltransferase 1 into COPI vesicles. Depletion of GOLPH3 altered the subcellular localization of these enzymes. In contrast, galactosyltransferase, an enzyme that does not interact with GOLPH3, was neither incorporated into COPI vesicles nor was dependent on GOLPH3 for proper localization. 相似文献
17.
Natasha A. Pereira Helen X. Pu Hazel Goh Zhiwei Song 《The Journal of biological chemistry》2014,289(21):14762-14770
GOLPH3 is a highly conserved protein found across the eukaryotic lineage. The yeast homolog, Vps74p, interacts with and maintains the Golgi localization of several mannosyltransferases, which is subsequently critical for N- and O-glycosylation in yeast. Through the use of a T7 phage display, we discovered a novel interaction between GOLPH3 and a mammalian glycosyltransferase, POMGnT1, which is involved in the O-mannosylation of α-dystroglycan. The cytoplasmic tail of POMGnT1 was found to be critical for mediating its interaction with GOLPH3. Loss of this interaction resulted in the inability of POMGnT1 to localize to the Golgi and reduced the functional glycosylation of α-dystroglycan. In addition, we showed that three clinically relevant mutations present in the stem domain of POMGnT1 mislocalized to the endoplasmic reticulum, highlighting the importance of identifying the molecular mechanisms responsible for Golgi localization of glycosyltransferases. Our findings reveal a novel role for GOLPH3 in mediating the Golgi localization of POMGnT1. 相似文献
18.
19.
《Biochemical and biophysical research communications》2013,433(3):338-344
Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in the development of several human cancers. However, the biological significance of GOLPH3 in glioma progression remains largely unknown. In this study, we report, for the first time, that downregulation of GOLPH3 led to clear reductions in glioma cell migration and invasion. In addition, downregulation of GOLPH3 inhibited the expression of the small GTPase RhoA as well as cytoskeletal reorganization, which are both required for glioma cell migration. Furthermore, we found that the observed reductions in glioma cell migration and RhoA level could be rescued by RhoA overexpression. Taken together, these results show that GOLPH3 contributes to the motility of glioma cells by regulating the expression of RhoA. 相似文献
20.