Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

Galen, Josse van; Campelo, Felix; Martínez‐Alonso, Emma; Scarpa, Margherita; Martı́nez-Menárguez, José A.; Malhotra, Vivek

doi:10.1083/jcb.201405009

Cited by 56 publications

(64 citation statements)

References 30 publications

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“…Our results show that Golgi cisternae curling starts already after 30 min of D-cer-C6 treatment, and that after 4 hr of treatment, virtually no flat stacks are observed (Figure 6C,D). In agreement with our previous analysis (van Galen et al, 2014), cisternae curling occurs towards the trans -Golgi cisternae/TGN, as monitored by the localization of specific late Golgi markers (Figure 6C). We extracted from these images the radius of flat cisternae,

r_{f l a t} = 510 \pm 21 n m

(average ± SEM; N = 22) and the radius of curling in the 4 hr D-cer-C6 treated Golgi cisternae,

R = 270 \pm 26 n m

(average ± SEM; N = 14), which are in agreement with the condition of area conservation (Equation A2).…”

Section: Resultssupporting

confidence: 93%

“…Our previously (van Galen et al, 2014) and presently reported ultrastructural data shows that normal Golgi morphology is altered in cells where SM metabolism had been disrupted. Under those conditions, the original flat-like cisternae curl into a concentric stacked onion-like structure.…”

Section: Methodsmentioning

confidence: 58%

“…Interestingly, we showed that these effects occur concomitantly with an overall reduction in the lateral lipid order of the Golgi membranes (Duran et al, 2012) as well as with striking alterations in the morphology of Golgi cisternae, which abandon their typical flat morphology and become highly curled (van Galen et al, 2014). Based on our findings, we suggested that short-chain SM might not be able to generate liquid-ordered nanodomains at the Golgi membranes (Duran et al, 2012).…”

Section: Introductionmentioning

confidence: 93%

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Sphingomyelin metabolism controls the shape and function of the Golgi cisternae

Campelo

Galen

Turacchio

et al. 2017

eLife

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The flat Golgi cisterna is a highly conserved feature of eukaryotic cells, but how is this morphology achieved and is it related to its function in cargo sorting and export? A physical model of cisterna morphology led us to propose that sphingomyelin (SM) metabolism at the trans-Golgi membranes in mammalian cells essentially controls the structural features of a Golgi cisterna by regulating its association to curvature-generating proteins. An experimental test of this hypothesis revealed that affecting SM homeostasis converted flat cisternae into highly curled membranes with a concomitant dissociation of membrane curvature-generating proteins. These data lend support to our hypothesis that SM metabolism controls the structural organization of a Golgi cisterna. Together with our previously presented role of SM in controlling the location of proteins involved in glycosylation and vesicle formation, our data reveal the significance of SM metabolism in the structural organization and function of Golgi cisternae.DOI: http://dx.doi.org/10.7554/eLife.24603.001

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r_{f l a t} = 510 \pm 21 n m

(average ± SEM; N = 22) and the radius of curling in the 4 hr D-cer-C6 treated Golgi cisternae,

R = 270 \pm 26 n m

(average ± SEM; N = 14), which are in agreement with the condition of area conservation (Equation A2).…”

Section: Resultssupporting

confidence: 93%

Section: Methodsmentioning

confidence: 58%

Section: Introductionmentioning

confidence: 93%

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Sphingomyelin metabolism controls the shape and function of the Golgi cisternae

Campelo

Galen

Turacchio

et al. 2017

eLife

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“…Van Galen and coll. recently showed modified organization of functional enzymatic domains and differential sorting of transmembrane proteins in the Trans-Golgi network after disruption of SM homeostasis [241]. …”

Section: Physiopathological Significancementioning

confidence: 99%

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

Carquin

D’Auria

Pollet

et al. 2016

Progress in Lipid Research

129

112

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The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicholson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decade, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs sec) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryotes to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution.

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“…This postulate is supported by results obtained with D-ceramide-C6. This lipid derivative causes the disruption of cholesterol-and sphingomyelin-enriched domains at the Golgi (63), which is accompanied by a swelling of cisternae and alterations in N-glycosylation (63,66), both strikingly coincidental with impairments at the Golgi occurring after actin depolymerization and pharmacological V-ATPase inhibition (49,57,58). Importantly, D-ceramide-C6 treatment also caused Golgi and TGN pH alkalization, which also occurs after actin-depolymerizing toxins (49).…”

Section: Table 2 Reduction Of Golgi Membrane Lipid Order Increases Gomentioning

confidence: 99%

Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex

Serra‐Peinado¹,

Sicart²,

Llopis³

et al. 2016

Journal of Biological Chemistry

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We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and the trans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H ؉ -translocating ATPase (V-ATPase), whose V 1 domain subunits B and C bind actin. We have generated a GFPtagged subunit B2 construct (GFP-B2) that is incorporated into the V 1 domain, which in turn is coupled to the V 0 sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V 1 -V 0 domains, which entails subunit B2 translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunits B2 and C1 and actin were detected. In addition, Golgi membrane lipid order disruption by D-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V 1 -V 0 domains of V-ATPase through the binding of microfilaments to subunits B and C and preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.The secretory pathway is characterized by progressive lumen acidification of its organelles, from almost neutral in the endoplasmic reticulum (ER) 4 (pH Ϸ7.1-7.2), along the cis-to-trans Golgi stack (pH Ϸ6.7-6.0), to more acidic in the trans-Golgi network (TGN) and secretory vesicles/granules (pH Ϸ5.0) (1-3). This pH gradient is crucial for post-translational modifications and membrane trafficking events (4, 5). The main molecular determinant of the progressive fall in pH along the secretory pathway is the vacuolar [H ϩ ]ATPase (V-ATPase) (6 -8). V-ATPase is a multisubunit complex composed of two large domains, V 0 and V 1 . The V 0 domain is a 260-kDa integral membrane complex made up of five different subunits (a, b, c, cЈ, cЉ, d, and e), which mediates proton translocation; the V 1 domain is a 600 -650-kDa peripheral complex composed of eight different subunits (A, B, C, D, E, F, G, and H), which is responsible for the ATP hydrolysis that provides the mechanical force necessary for proton (H ϩ ) translocation (7, 9 -11). Whereas they are the primary source of proton delivery to endomembranes consuming ATP, the final steady-state pH in the secretory pathway is the result of the balance between active H ϩ pumping by the V-ATPase, passive H ϩ efflux through organelle endogenous H ϩ permeability, and differences in counter-ion conductance (3, 12).How differences in the pH of individual secretory compartments are generated is not well understood. Differential VATPase density and/or local regulatory mechanisms in secretory organelles and subcompartments are possible (13). In this respect, V-ATPase-dependent proton translocation could be regulated by several mechanisms, which include the following: (a) differential V-ATPase subunit expression; (b) intracel...

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Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

Abstract: Sphingomyelin-mediated organization of resident transmembrane proteins into specific membrane domains at the trans-Golgi network is necessary for normal enzymatic activity.

Cited by 56 publications

References 30 publications

Sphingomyelin metabolism controls the shape and function of the Golgi cisternae

Sphingomyelin metabolism controls the shape and function of the Golgi cisternae

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex

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