GRASP55 regulates intra‐Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis
2021; Springer Nature; Volume: 40; Issue: 20 Linguagem: Inglês
10.15252/embj.2021107766
ISSN1460-2075
AutoresPrathyush Pothukuchi, Ilenia Agliarulo, Marinella Pirozzi, Riccardo Rizzo, Domenico Russo, Gabriele Turacchio, Julian Nüchel, Jia‐Shu Yang, Charlotte Gehin, Laura Capolupo, María José Hernandez‐Corbacho, Ansuman Biswas, Giovanna Vanacore, Nina Dathan, Takahiro Nitta, Petra Henklein, Mukund Thattai, Jin‐ichi Inokuchi, Victor W. Hsu, Markus Plomann, Lina M. Obeid, Yusuf A. Hannun, Alberto Luini, Giovanni D’Angelo, Seetharaman Parashuraman,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle13 September 2021Open Access Source DataTransparent process GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis Prathyush Pothukuchi Prathyush Pothukuchi orcid.org/0000-0002-6242-2319 Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Ilenia Agliarulo Ilenia Agliarulo Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy These authors contributed equally to this work Search for more papers by this author Marinella Pirozzi Marinella Pirozzi Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy These authors contributed equally to this work Search for more papers by this author Riccardo Rizzo Riccardo Rizzo Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Domenico Russo Domenico Russo Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Gabriele Turacchio Gabriele Turacchio Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Julian Nüchel Julian Nüchel orcid.org/0000-0002-8126-415X Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany Search for more papers by this author Jia-Shu Yang Jia-Shu Yang Division of Rheumatology, Inflammation and Immunity, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Charlotte Gehin Charlotte Gehin orcid.org/0000-0003-1164-2968 École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Laura Capolupo Laura Capolupo École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Maria Jose Hernandez-Corbacho Maria Jose Hernandez-Corbacho Stony Brook University Medical Center, Stony Brook, NY, USA Search for more papers by this author Ansuman Biswas Ansuman Biswas National Center of Biological Sciences, Bengaluru, India Search for more papers by this author Giovanna Vanacore Giovanna Vanacore Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Nina Dathan Nina Dathan Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Takahiro Nitta Takahiro Nitta Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Petra Henklein Petra Henklein Universitätsmedizin Berlin Institut für Biochemie Charité CrossOver Charitéplatz 1 / Sitz, Berlin, Germany Search for more papers by this author Mukund Thattai Mukund Thattai National Center of Biological Sciences, Bengaluru, India Search for more papers by this author Jin-Ichi Inokuchi Jin-Ichi Inokuchi orcid.org/0000-0002-0703-5746 Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Victor W Hsu Victor W Hsu orcid.org/0000-0002-6763-4636 Division of Rheumatology, Inflammation and Immunity, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Markus Plomann Markus Plomann orcid.org/0000-0001-6509-5627 Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany Search for more papers by this author Lina M Obeid Lina M Obeid orcid.org/0000-0002-0734-0847 Stony Brook University Medical Center, Stony Brook, NY, USA Deceased. Search for more papers by this author Yusuf A Hannun Yusuf A Hannun Stony Brook University Medical Center, Stony Brook, NY, USA Search for more papers by this author Alberto Luini Alberto Luini Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Giovanni D'Angelo Giovanni D'Angelo orcid.org/0000-0002-0734-4127 Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Seetharaman Parashuraman Corresponding Author Seetharaman Parashuraman [email protected] orcid.org/0000-0001-5113-4592 Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Prathyush Pothukuchi Prathyush Pothukuchi orcid.org/0000-0002-6242-2319 Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Ilenia Agliarulo Ilenia Agliarulo Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy These authors contributed equally to this work Search for more papers by this author Marinella Pirozzi Marinella Pirozzi Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy These authors contributed equally to this work Search for more papers by this author Riccardo Rizzo Riccardo Rizzo Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Domenico Russo Domenico Russo Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Gabriele Turacchio Gabriele Turacchio Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Julian Nüchel Julian Nüchel orcid.org/0000-0002-8126-415X Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany Search for more papers by this author Jia-Shu Yang Jia-Shu Yang Division of Rheumatology, Inflammation and Immunity, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Charlotte Gehin Charlotte Gehin orcid.org/0000-0003-1164-2968 École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Laura Capolupo Laura Capolupo École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Maria Jose Hernandez-Corbacho Maria Jose Hernandez-Corbacho Stony Brook University Medical Center, Stony Brook, NY, USA Search for more papers by this author Ansuman Biswas Ansuman Biswas National Center of Biological Sciences, Bengaluru, India Search for more papers by this author Giovanna Vanacore Giovanna Vanacore Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Nina Dathan Nina Dathan Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Takahiro Nitta Takahiro Nitta Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Petra Henklein Petra Henklein Universitätsmedizin Berlin Institut für Biochemie Charité CrossOver Charitéplatz 1 / Sitz, Berlin, Germany Search for more papers by this author Mukund Thattai Mukund Thattai National Center of Biological Sciences, Bengaluru, India Search for more papers by this author Jin-Ichi Inokuchi Jin-Ichi Inokuchi orcid.org/0000-0002-0703-5746 Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Victor W Hsu Victor W Hsu orcid.org/0000-0002-6763-4636 Division of Rheumatology, Inflammation and Immunity, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Markus Plomann Markus Plomann orcid.org/0000-0001-6509-5627 Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany Search for more papers by this author Lina M Obeid Lina M Obeid orcid.org/0000-0002-0734-0847 Stony Brook University Medical Center, Stony Brook, NY, USA Deceased. Search for more papers by this author Yusuf A Hannun Yusuf A Hannun Stony Brook University Medical Center, Stony Brook, NY, USA Search for more papers by this author Alberto Luini Alberto Luini Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Giovanni D'Angelo Giovanni D'Angelo orcid.org/0000-0002-0734-4127 Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Seetharaman Parashuraman Corresponding Author Seetharaman Parashuraman [email protected] orcid.org/0000-0001-5113-4592 Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy Search for more papers by this author Author Information Prathyush Pothukuchi1, Ilenia Agliarulo1, Marinella Pirozzi1, Riccardo Rizzo1,9, Domenico Russo1, Gabriele Turacchio1, Julian Nüchel2, Jia-Shu Yang3, Charlotte Gehin4, Laura Capolupo4, Maria Jose Hernandez-Corbacho5, Ansuman Biswas6, Giovanna Vanacore1, Nina Dathan1, Takahiro Nitta7, Petra Henklein8, Mukund Thattai6, Jin-Ichi Inokuchi7, Victor W Hsu3, Markus Plomann2, Lina M Obeid5, Yusuf A Hannun5, Alberto Luini1, Giovanni D'Angelo1,4 and Seetharaman Parashuraman *,1 1Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy 2Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany 3Division of Rheumatology, Inflammation and Immunity, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA 4École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 5Stony Brook University Medical Center, Stony Brook, NY, USA 6National Center of Biological Sciences, Bengaluru, India 7Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan 8Universitätsmedizin Berlin Institut für Biochemie Charité CrossOver Charitéplatz 1 / Sitz, Berlin, Germany 9Present address: Institute of Nanotechnology, National Research Council (CNR-NANOTEC), Lecce, Italy *Corresponding author. Tel: +39-081-6132283; E-mail: [email protected] The EMBO Journal (2021)40:e107766https://doi.org/10.15252/embj.2021107766 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis. SYNOPSIS How the compartmentalized localization of glycosylation enzymes in Golgi is achieved and how it regulates glycosylation is incompletely understood. Here, GRASP55 is found to control competition between glucosylceramide synthase (GCS) and sphingomyelin synthase 1 (SMS1), two enzymes of the glycosphingolipid (GSL) biosynthetic pathway, by regulating trans-Golgi localization of GCS. SMS1 and GCS localize to the trans-Golgi, where they compete for the shared substrate ceramide. GRASP55 binding to the C-terminus of GCS prevents its sorting into retrograde COPI vesicles, thus localizing GCS to the trans-Golgi. Absence of GRASP55-GCS interaction relocates GCS to the cis-Golgi, while SMS1 remains in the trans-Golgi. GCS in the cis-Golgi may have preferential access to ceramide compared to SMS1. Preferential GCS access results in increased glycosphingolipid biosynthesis in the absence of GRASP55. Introduction Glycans are one of the fundamental building blocks of the cell and play key roles in development and physiology (Bishop et al, 2007; Kohyama-Koganeya et al, 2011; Ryczko et al, 2016; Varki, 2017; Akintayo & Stanley, 2019). Cellular glycan profiles are sensitive to changes in cell state and/or differentiation and are also important contributors to the process (Russo et al, 2018b). Indeed, several developmental disorders are associated with impaired production of glycans (Chang et al, 2018). Thus, how the glycan biosynthesis is regulated to achieve specific cellular glycan profiles is an important biological problem. In eukaryotes, glycans are assembled mainly by the Golgi apparatus on cargo proteins and lipids that traverse the organelle (Stanley, 2011). Glycan biosynthesis happens in a template-independent fashion (Varki & Kornfeld, 2015), yet the products are not random polymers of sugars but a defined distribution of glycans that is cell-type and cargo-specific (Rudd et al, 2015; Varki & Kornfeld, 2015). This suggests that their biosynthesis is guided by regulated program(s). Transcriptional programs have been identified that contribute to defining the glycome of a cell, but they only partially account for it (Nairn et al, 2008, 2012; Varki & Kornfeld, 2015). An obviously important but unexplored factor that influences glycosylation is the Golgi apparatus itself (Varki, 1998; Maccioni et al, 2002). The Golgi apparatus is a central organelle of the secretory pathway that processes newly synthesized cargoes coming from the endoplasmic reticulum (ER), primarily by glycosylation, before sorting them toward their correct destination in the cell. It consists of a stack of 4–11 cisternae (Klumperman, 2011), populated by enzymes and accessory proteins that maintain a suitable milieu for the enzymes to act on biosynthetic cargoes. The stack is polarized with a cis-side where cargoes arrive and a trans-side from where they leave. The enzymes are not homogeneously distributed across the Golgi stack but are restricted or compartmentalized to 1–3 specific cisternae. The cisternal maturation model provides a conceptual framework for understanding Golgi enzyme compartmentalization (Nakano & Luini, 2010; Glick & Luini, 2011). According to the model, secretory cargoes are transported forward by the anterograde flux mediated by cisternal progression, which consists of constant formation and consumption of cis and trans cisternae, respectively. The retention of Golgi glycosylation enzymes in the face of this continuous forward flux is mediated by their retrograde transport that acts as counterbalance for the forward transport. The retrograde transport is promoted by coat protein complex I (COPI) machinery (Rabouille & Klumperman, 2005; Popoff et al, 2011; Papanikou et al, 2015; Ishii et al, 2016; Liu et al, 2018) and is assisted in this process by adaptor molecules like GOLPH3 (Tu et al, 2008, 2012; preprint: Rizzo et al, 2019), conserved oligomeric complex (COG) proteins, and Golgi matrix proteins especially Golgins (Eckert et al, 2014; Wong & Munro, 2014; Blackburn et al, 2019). However, the specific molecular mechanisms and processes by which the same retrograde transport pathway promotes localization of enzymes to distinct cisternae remain unknown. The compartmentalized localization of enzymes has been suggested to influence both sequential as well as competing glycosylation reactions. The localization of enzymes along the cis-trans axis reflecting their order of action (Dunphy & Rothman, 1985) has been suggested to influence the efficiency of sequential processing reactions (Fisher et al, 2019). On the other hand, the promiscuity of glycosylation enzymes (Biswas & Thattai, 2020) makes compartmentalized localization of competing enzymes a critical factor in determining the specificity in glycan output (i.e., the type and quantity of glycans produced) (Dunphy & Rothman, 1985; Pothukuchi et al, 2019; Jaiman & Thattai, 2020). When two or more enzymes compete for a substrate, the order in which they get access to it can substantially influence the glycans produced and subsequently the physiological outcomes. Competing reactions are frequent in glycosylation pathways, and all known glycosylation pathways have one or more competing glycosylation steps. Nevertheless, how the compartmentalized localization of competing enzymes is achieved, how it is regulated to influence glycosylation reactions, and what the physiological relevance of this regulation is remain unexplored. To evaluate and understand the contribution of Golgi compartmentalization in regulating glycosylation, we have focused our study on sphingolipid (SL) glycosylation. We chose this model system for several reasons: a. It is well characterized from both biochemical and transcriptional perspectives (Halter et al, 2007; D'Angelo et al, 2013; Russo et al, 2018b); b. the glycosylation reaction is less influenced by the cargo structure in contrast to protein glycosylation and thus is a cleaner system to study effects of Golgi processes on glycosylation; c. there are simple biochemical methods available to analyze SL glycosylation (D'Angelo et al, 2013); and d. finally, SLs have important roles in physiology and development (Hannun & Obeid, 2018; Russo et al, 2018a). The SL glycosylation pathway exhibits the essential features of glycosylation pathways like localization of enzymes reflecting their order of action and also at least two competing reaction steps that are important in determining the metabolic outcome of the pathway (see below). Further, while enzymes of the pathway are well characterized, molecular players regulating their sub-Golgi compartmentalization are unknown. By studying SL glycosylation, we identify GRASP55 as an important factor that compartmentalizes two enzymes catalyzing critical branch points of the SL glycosylation pathway. GRASP55 binds to and prevents the entry of these enzymes into retrograde transport carriers. This retaining action of GRASP55 is essential for dynamic compartmentalization of these enzymes in the Golgi stack. The competing enzymes thus positioned at appropriate levels in the Golgi stack regulate cargo flux across competing reactions of the pathway and determine the metabolic outcome viz. sphingolipid produced by the cell. These results delineate a molecular mechanism of enzyme compartmentalization and how it controls cell surface glycan profile. Results Disruption of Golgi organization alters SL biosynthesis SL biosynthesis starts with the production of ceramide (Cer) in the ER, which is then processed in the Golgi to sphingomyelin (SM) or glycosphingolipids (GSLs). The model cell system we use, HeLa cells, produces two species of GSLs—globosides (Gb3) and gangliosides (GM1 and GM3) (Halter et al, 2007; D'Angelo et al, 2013; Russo et al, 2018b) (See Fig 1A for schematic of the SL system in HeLa cells). This SL pathway includes sequential processing of Cer to complex GSLs as well as two bifurcating steps where the substrates get differentially channeled. The first is the bifurcation between SM and glucosylceramide (GlcCer) biosynthesis, where the substrate Cer is channeled into either of the pathways. The second is the biosynthesis of Gb3 or GM3 from lactosylceramide (LacCer). These two critical steps determine the amount and type of SLs produced by the cell. We first examined the localization of SL biosynthetic enzymes and found that they localize to three distinct zones in the secretory pathway (Fig 1B, Appendix Fig S1): (i) the early secretory pathway including the ER and the cis/medial-Golgi (C1, C2 cisternae), where Cer biosynthetic enzymes are localized (33), have little if any SL biosynthetic enzymes except for a slightly elevated amount of GM3S and GlcCer synthase (GCS) in the cis/medial-Golgi compared with other GSL biosynthetic enzymes; (ii) medial/trans-Golgi (C3, C4 cisternae) where most of the GSL biosynthetic enzymes are present alongside substantial amounts of Sphingomyelin synthase 1 (SMS1) and (iii) trans-Golgi network (TGN), where SMS1 predominates. While all the GSL biosynthetic enzymes show a gradient of increasing concentration from cis- to trans-Golgi, the gradient is much sharper in the case of GB3S and LacCer synthase (LCS) compared with GCS and GM3S (Appendix Fig S1). Thus, the SL biosynthetic enzymes are distributed reflecting their order of action with precursor (Cer) producing enzymes in the early secretory pathway and the Cer processing enzymes in late secretory pathway, which is in turn divided into two distinct zones where GSL and SM biosynthesis predominate. Of note, we expressed HA-tagged enzymes (see Materials and Methods) for our studies since the endogenous enzymes were barely detectable and efficient antibodies for EM studies of endogenous enzymes were not available. Nevertheless, the localization mostly reflects expected localization based on enzyme activity and previously published evidence (Parashuraman & D'Angelo, 2019). A notable exception is the localization of GCS that was shown to be on the cis-side of the Golgi (Halter et al, 2007) contrary to what we report here. This is because the earlier studies had used a construct with a tag that blocks the signal for intra-Golgi localization that we identify and describe here. When this signal is blocked, localization of GCS is altered resulting in localization to cis-Golgi (see below). Figure 1. Disruption of SL biosynthetic machinery organization alters SL output A. Schematic representation of GSL biosynthetic pathway in HeLa cells (Glu, glucose; Gal, galactose; Sia, N-acetylneuraminic acid; Cer, ceramide). Products of biosynthesis are represented in bold and enzymes that catalyze the reactions in gray. The arrows represent the SL metabolic flux from ceramide. B. Schematic representation of GSL biosynthetic zones in HeLa, SM biosynthesis predominates in TGN, whereas GSL and SM productions happen in medial/trans-Golgi (C3 and C4 cisternae). Cis-Golgi/ER is where Ceramide biosynthesis happens with little, if any, SL production. CerS* refers to the group of Ceramide synthases localized to the ER. The size of the lipid label arbitrarily represents the proportion of the lipid expected to be synthesized in the compartment based on the localization of corresponding enzymes. C. High-performance thin-layer chromatography (HPTLC) profile of HeLa cells pulsed for 2 h with [3H]-sphingosine and chased for 24 h. The peaks corresponding to each SL species are indicated, and numbers represent each SL species as percentage of total SL. D. The total radioactivity associated with Cer, SM, and GSLs (GluCer, LacCer, Gb, and GM), or GM and Gb were quantified and presented as percentages relative to total. Data represented as means ± SD of three independent experiments. E, F. Biosynthesis of SL in HeLa cells expressing GTP-locked mutants of Sar1 or ARF1 or treated with Brefeldin A (BFA; 5 μg/ml) was measured by [3H]-sphingosine pulse-chase assay. Radioactivity associated with GSLs was quantified and represented as fold change with respect to control. (E) For BFA-treated cells, the SL output was measured 8 h after pulse. Data represented as means ± SD of two independent experiments. *P < 0.05, **P < 0.01 (Student's t-test). (F) The ratio of GM/Gb is represented. Data represented as means ± SD of two independent experiments. *P < 0.05, **P < 0.01 (Student's t-test). Download figure Download PowerPoint Next, SL output of this system was measured by metabolic labeling with 3H-sphingosine, a precursor of ceramide. This revealed the following distribution of products at quasi steady state i.e., 24 h after labeling: SM (70%), globosides (10%), and gangliosides (5%) and rest remaining as precursors (Cer, GlcCer or LacCer; 15%) (Fig 1C and D). The GSLs (globosides, gangliosides, and GSL precursors GlcCer and LacCer) together constituted 25% of total SLs produced. We will refer to the ratio of GSL:SM::25:70 as SL output and the ratio of gangliosides (GM) to Globosides (Gb), GM:Gb::5:10 as GSL output (Fig 1D). For simplicity, the SL output will be represented as GSL fraction since a change in GSLs is always associated with a proportional change in SM in the opposite direction. For GSL output, the situation is complex since a substantial portion of signal remains as precursors (GlcCer and LacCer), and so GSL output will be represented as a GM/Gb ratio which under the control conditions corresponds to 0.5 (GM:Gb::5:10). To summarize, the SL machinery has a compartmentalized localization across the Golgi in HeLa cells and produces a SL output such that 70% of the Cer is directed toward the production of SM and 25% toward the production of GSLs. Within this 25, 5% is directed toward the production of gangliosides and 10% toward the production of globosides. This distribution of glycoforms produced by the Golgi apparatus has largely been ascribed to the expression of the corresponding glycosylation enzymes (Maccioni et al, 2002; Nairn et al, 2008, 2012). To assess the contribution of enzyme compartmentalization to this, we monitored SL output after disrupting the spatial organization of SL biosynthetic enzymes by a) overexpressing GTP-locked mutants of monomeric GTPases——secretion-associated Ras-related GTPase (Sar1 H79G) and ADP ribosylation factor 1 (ARF1 Q71L) that are well known to disorganize the secretory pathway (Zhang et al, 1994; Aridor et al, 1995) and b) by treating the cells with Brefeldin A, which causes relocation of Golgi enzymes back to the ER. Overexpression of Sar1 H79G led to collapse of the Golgi apparatus into the ER with SL biosynthetic enzymes showing a reticular ER pattern (Appendix Fig S2A). On the other hand, overexpression of ARF1 Q71L mutant led to disruption of stacked cisternal structure of the Golgi, which was replaced by tubulo-vesicular clusters (Appendix Fig S2B), with no separation between cis- and trans-Golgi markers (Appendix Fig S2C) (List of recombinant DNA used in this study are listed in Appendix Table S2). The treatment with Brefeldin A led to the translocation of the enzymes back into the ER as expected, apart from SMS1 which while present in the ER also displayed presence in some punctate structures (Appendix Fig S2A). The SL output was altered in these cells, and consistently, in all three conditions there was an increased production of GSLs over SM and gangliosides over globosides (Appendix Fig S2D and E). The SL output represented as fold change in GSL fraction showed that GSL production in these cells increased by 1.5–1.9 fold over control cells (Fig 1E). Similarly, GSL output measured as GM/Gb ratio changed from 0.5 in control cells to 1.3–1.5 in treated cells (Fig 1F). These data suggest that impaired spatial organization of enzymes correlates with altered SL output, and especially, the output from steps involving competing reactions is sensitive to disorganization of the Golgi. The contribution of enzyme expression to determination of glycosylation is well established (Nairn et al, 2012) but the contribution of the Golgi organization and its importance to this process was not clear. These results underscore a significant and substantive role played by the Golgi apparatus in determining the glycan output of a cell. GRASP55 regulates SL output by controlling substrate flux between competing glycosylation pathways Given the importance of the organization of the Golgi apparatus, and likely of the SL biosynthetic machinery localized to the organelle, to determining SL output, we wanted to identify the molecular players involved in this process. Retention of enzymes in the Golgi depends on their COPI-dependent retrograde transport. Golgi matrix proteins especially Golgins contribute to specificity in this process (Wong & Munro, 2014) and thus to compartmentalization of enzymes. So, to identify specific regulators of compartmentalization of SL biosynthetic enzymes, we systematically silenced Golgi matrix proteins and studied the effect on SL production. Among the 14 matrix proteins tested by depletion, downregulation of GRASP55 significantly increased the production of GSLs (a 40% increase in GSLs compared with control) while downregulation of GOPC and GCC2 led to a decrease in GSL levels (Fig 2A) (siRNA sequences used in this study to downregulate indicated human gene expression are listed in Appendix Table S3). We followed up on GRASP55 since its depletion altered SL output similar to that obtained by disorganization of the Golgi apparatus (Fig 1E). Figure 2. GRASP55 regulates SL biosynthesis A. HeLa cells were treated with control or indicated siRNA (pool of 4 or 2 as indicated in me
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