Sphingolipid metabolic flow controls phosphoinositide turnover at the trans Golgi network

AbstractSphingolipids are membrane lipids, which are globally required for eukaryotic life. Sphingolipid composition varies among endomembranes with pre- and post-Golgi compartments being poor and rich in sphingolipids, respectively. Thanks to this different sphingolipid content, pre- and post-Golgi membranes serve different cellular functions. Nevertheless, how subcellular sphingolipid levels are maintained in spite of trafficking and metabolic fluxes is only partially understood. Here we describe a homeostatic control circuit that controls sphingolipid levels at the trans Golgi network. Specifically, we show that sphingomyelin production at the trans Golgi network triggers a signalling reaction leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol, and sphingolipid transport to the trans Golgi network, PtdIns(4)P consumption leads to the interruption of this transport in response to excessive sphingomyelin production. Based on this evidence we envisage a model where this homeostatic circuit maintains the sphingolipid composition of trans Golgi network and thus of post-Golgi compartments constant, against instant fluctuations in the sphingolipid biosynthetic flow.

Download Full-text

p230 is associated with vesicles budding from the trans-Golgi network

Journal of Cell Science ◽

10.1242/jcs.109.12.2811 ◽

1996 ◽

Vol 109 (12) ◽

pp. 2811-2821 ◽

Cited By ~ 7

Author(s):

P.A. Gleeson ◽

T.J. Anderson ◽

J.L. Stow ◽

G. Griffiths ◽

B.H. Toh ◽

...

Keyword(s):

Brefeldin A ◽

Immunogold Labeling ◽

Coated Vesicles ◽

Specific Marker ◽

Very High Frequency ◽

Trans Golgi Network ◽

Golgi Membranes ◽

Vesicle Biogenesis ◽

Heptad Repeats ◽

Golgi Network

Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTP gamma S or AlF4- resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (approximately 10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.

Download Full-text

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

The Journal of Cell Biology ◽

10.1083/jcb.200901145 ◽

2009 ◽

Vol 185 (4) ◽

pp. 601-612 ◽

Cited By ~ 271

Author(s):

Robin W. Klemm ◽

Christer S. Ejsing ◽

Michal A. Surma ◽

Hermann-Josef Kaiser ◽

Mathias J. Gerl ◽

...

Keyword(s):

Membrane Trafficking ◽

Secretory Pathway ◽

Membrane Lipids ◽

Secretory Vesicles ◽

Shotgun Lipidomics ◽

Yeast Saccharomyces Cerevisiae ◽

Trans Golgi Network ◽

Sorting Station ◽

Golgi Network ◽

First Time

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.

Download Full-text

Arf3 Is Activated Uniquely at the trans-Golgi Network by Brefeldin A-inhibited Guanine Nucleotide Exchange Factors

Molecular Biology of the Cell ◽

10.1091/mbc.e10-01-0016 ◽

2010 ◽

Vol 21 (11) ◽

pp. 1836-1849 ◽

Cited By ~ 27

Author(s):

Florin Manolea ◽

Justin Chun ◽

David W. Chen ◽

Ian Clarke ◽

Nathan Summerfeldt ◽

...

Keyword(s):

Guanine Nucleotide Exchange Factors ◽

Temperature Sensitive ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Membrane Composition ◽

Trans Golgi Network ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Golgi Membranes ◽

Golgi Network

It is widely assumed that class I and II Arfs function interchangeably throughout the Golgi complex. However, we report here that in vivo, Arf3 displays several unexpected properties. Unlike other Golgi-localized Arfs, Arf3 associates selectively with membranes of the trans-Golgi network (TGN) in a manner that is both temperature-sensitive and uniquely dependent on guanine nucleotide exchange factors of the BIGs family. For example, BIGs knockdown redistributed Arf3 but not Arf1 from Golgi membranes. Furthermore, shifting temperature to 20°C, a temperature known to block cargo in the TGN, selectively redistributed Arf3 from Golgi membranes. Arf3 redistribution occurred slowly, suggesting it resulted from a change in membrane composition. Arf3 knockdown and overexpression experiments suggest that redistribution is not responsible for the 20°C block. To investigate in more detail the mechanism for Arf3 recruitment and temperature-dependent release, we characterized several mutant forms of Arf3. This analysis demonstrated that those properties are readily separated and depend on pairs of residues present at opposite ends of the protein. Furthermore, phylogenetic analysis established that all four critical residues were absolutely conserved and unique to Arf3. These results suggest that Arf3 plays a unique function at the TGN that likely involves recruitment by a specific receptor.

Download Full-text

Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network

The Journal of Cell Biology ◽

10.1083/jcb.200908040 ◽

2009 ◽

Vol 187 (7) ◽

pp. 1055-1069 ◽

Cited By ~ 77

Author(s):

Julia von Blume ◽

Juan M. Duran ◽

Elena Forlanelli ◽

Anne-Marie Alleaume ◽

Mikhail Egorov ◽

...

Keyword(s):

Mammalian Cells ◽

Isotope Labeling ◽

Secretory Protein ◽

Protein Profiling ◽

Secretory Proteins ◽

Actin Remodeling ◽

Trans Golgi Network ◽

Culture Cells ◽

Golgi Membranes ◽

Golgi Network

Knockdown of the actin-severing protein actin-depolymerizing factor (ADF)/cofilin inhibited export of an exogenously expressed soluble secretory protein from Golgi membranes in Drosophila melanogaster and mammalian tissue culture cells. A stable isotope labeling by amino acids in cell culture mass spectrometry–based protein profiling revealed that a large number of endogenous secretory proteins in mammalian cells were not secreted upon ADF/cofilin knockdown. Although many secretory proteins were retained, a Golgi-resident protein and a lysosomal hydrolase were aberrantly secreted upon ADF/cofilin knockdown. Overall, our findings indicate that inactivation of ADF/cofilin perturbed the sorting of a subset of both soluble and integral membrane proteins at the trans-Golgi network (TGN). We suggest that ADF/cofilin-dependent actin trimming generates a sorting domain at the TGN, which filters secretory cargo for export, and that uncontrolled growth of this domain causes missorting of proteins. This type of actin-dependent compartmentalization and filtering of secretory cargo at the TGN by ADF/cofilin could explain sorting of proteins that are destined to the cell surface.

Download Full-text