Selective Roles for Cholesterol and Actin in Compartmentalization of Different Proteins in the Golgi and Plasma Membrane of Polarized Cells

Lebreton, Stéphanie; Paladino, Simona; Zurzolo, Chiara

doi:10.1074/jbc.m803819200

Cited by 37 publications

(69 citation statements)

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“…Consistent with this hypothesis, we had previously shown that a double point mutation in the GFP sequence (S49/71) that prevents GFP dimerization also impairs oligomerization in the Golgi and the consequent apical sorting of the protein 11 . Taken together these data indicate that also in the Golgi of epithelial cells the capacity of dimerization is required for the construction of cholesterol-dependent higher order molecular organization of GPI-APs 26,27 .…”

Section: Discussionmentioning

confidence: 54%

“…Because in polarized MDCK cells cholesterol is a key regulator of the Golgi homo-clustering of apical GPI-APs 11,26,27 we asked whether differences in the cellular content of cholesterol between polarized and non-polarized cells could explain the different organization of GPI-APs at the cell surface. To address this we analyzed the B of GFP-FR in non-polarized MDCK cells following exogenous addition of cholesterol ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Golgi sorting regulates organization and activity of GPI proteins at apical membranes

et al. 2014

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Here, we combined classical biochemistry with novel biophysical approaches to study with high spatial and temporal resolution the organization of GPI-anchored proteins (GPI-APs) at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, following sorting in the Golgi, each GPI-AP reaches the apical surface in homo-clusters. Golgi-derived homo-clusters are required for their subsequent plasma membrane organization into cholesterol-dependent hetero-clusters. By contrast, in non-polarized MDCK cells GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form hetero-clusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, different from fibroblasts, in polarized epithelial cells a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and the function of GPI-APs at the apical surface.

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Section: Discussionmentioning

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Golgi sorting regulates organization and activity of GPI proteins at apical membranes

et al. 2014

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“…These data can be explained in at least two different ways: Either, the addition of cholesterol stabilises the interaction between the basolateral GPI-AP and lipid rafts (i.e. by rigidifying the raft membranes or interfering with the free diffusion of proteins) (Lebreton et al, 2008), thus allowing the stabilisation of the protein in rafts and consequently its oligomerisation and apical sorting; or, cholesterol addition may change the characteristics of the surrounding lipid environment (i.e. conversion of basolateral rafts to apical rafts), thus enabling the protein originally associated with the basolateral raft to oligomerise and to be delivered apically.…”

Section: Addition Of Cholesterol Results In Gfp-prp Oligomerisation Amentioning

confidence: 99%

Different GPI-attachment signals affect the oligomerisation of GPI-anchored proteins and their apical sorting

Paladino

Lebreton

Tivodar

et al. 2008

Journal of Cell Science

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To understand the mechanism involved in the apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) we fused to the C-terminus of GFP the GPI-anchor-attachment signal of the folate receptor (FR) or of the prion protein (PrP), two native GPI-anchored proteins that are sorted apically or basolaterally, respectively, in MDCK cells. We investigated the behaviour of the resulting fusion proteins GFP-FR and GFP-PrP by analysing three parameters: their association with DRMs, their oligomerisation and their apical sorting. Strikingly, we found that different GPI-attachment signals differently modulate the ability of the resulting GFP-fusion protein to oligomerise and to be apically sorted. This is probably owing to differences in the GPI anchor and/or in the surrounding lipid microenvironment. Accordingly, we show that addition of cholesterol to the cells is necessary and sufficient to drive the oligomerisation and consequent apical sorting of GFP-PrP, which under control conditions does not oligomerise and is basolaterally sorted.

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“…associated proteins ( 45 ). However, it is not known how factors, such as membrane domain heterogeneity, TGN membrane continuity, or possible changes to PI4KII ␣ lateral diffusion, might contribute to the cholesterolsensitivity of the enzyme.…”

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confidence: 99%

Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIα at the trans-Golgi network

Minogue

Chu

Westover

et al. 2010

Journal of Lipid Research

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Phosphatidylinositol 4-phosphate (PI4P) is generated by phosphorylation of phosphatidylinositol (PI) on the 4-position by phosphatidylinositol 4-kinases ( 1 ). In mammalian cells ( 2, 3 ), PI4P synthesis is predominately accounted for by the type II phosphatidylinositol 4-kinase II ␣ isoform (PI4KII ␣ ). Our most recent work has shown that loss of PI4KII ␣ activity in mice leads to late-onset neurodegeneration ( 2 ). In addition, Li et al. ( 4 ) have demonstrated that PI4KII ␣ is overexpressed in a wide range of common cancers where it has a key role in promoting angiogenesis. However, despite its emerging importance in disease, little is known about endogenous factors that regulate PI4KII ␣ , and there are currently no pharmacological reagents available to modulate its activity in cells. We previously demonstrated that PI4KII ␣ activity is sensitive to membrane cholesterol levels ( 5 ). Given the signifi cant role of the enzyme in major pathologies, we sought to investigate the biochemical and biophysical mechanisms that underlie sterol-sensitive PI4P synthesis.One possible mechanism through which cholesterol could affect PI4KII ␣ is by modulating its membrane mobility ( 6-10 ). Lateral diffusion of a membrane-associated protein determines its interaction dynamics with other membrane components (reviewed in Ref. 6 ), and in the case of an enzyme such as PI4KII ␣ , this is likely to be important for the kinetics of product formation. However, it is important to point out that nothing is known Abstract Type II phosphatidylinositol 4-kinase II ␣ (PI4KII ␣ ) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular traffi cking. Recently PI4KII ␣ has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KII ␣ mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KII ␣ activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KII ␣ to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fl uorescent protein (eGFP)-tagged PI4KII ␣ demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KII ␣ was assessed by fl uorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KII ␣ . Further measurements revealed that the reduction in the mobile fraction of PI4KII ␣ correlated with a loss of trans -Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme...

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Selective Roles for Cholesterol and Actin in Compartmentalization of Different Proteins in the Golgi and Plasma Membrane of Polarized Cells

Cited by 37 publications

References 50 publications

Golgi sorting regulates organization and activity of GPI proteins at apical membranes

Golgi sorting regulates organization and activity of GPI proteins at apical membranes

Different GPI-attachment signals affect the oligomerisation of GPI-anchored proteins and their apical sorting

Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIα at the trans-Golgi network

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