HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/7/866 03-0782fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ROBENEK, M. J. Articles by ROBENEK, H. Search for Related Content PubMed PubMed Citation Articles by ROBENEK, M. J. Articles by ROBENEK, H.

Lipids partition caveolin-1 from ER membranes into lipid droplets: updating the model of lipid droplet biogenesis^{1 ,2}

MIRKO J. ROBENEK^*, NICHOLAS J. SEVERS, KARIN SCHLATTMANN, GABRIELE PLENZ, KLAUS-PETER ZIMMER^*, DAVID TROYER and HORST ROBENEK^,3

^* University Children’s Hospital, and Departments of
Cell Biology and Ultrastructure Research, Institute for Arteriosclerosis Research, and
Cardiology and Angiology, and Thoracic and Cardiovascular Surgery, University of Münster, Germany; and
Imperial College London, National Heart and Lung Institute, London, UK

³Correspondence: Department of Cell Biology and Ultrastructure Research, Institute for Arteriosclerosis Research, University of Münster, Domagkstr. 3, 48149 Münster, Germany. E-mail: robenek{at}uni-muenster.de

SPECIFIC AIMS

The current model of lipid droplet formation assumes caveolin-1 resides in the cytoplasmic membrane leaflet of the endoplasmic reticulum (ER) and is transferred along with this leaflet to the surface of the nascent lipid droplet—but not to its core—as neutral lipids inundate between the membrane leaflets, causing the cytoplasmic leaflet to bulge and the droplet to bud off the ER. We explored how caveolin-1 accesses lipid droplets from the ER by localizing caveolin-1 in ER membranes and in lipid droplets of cultured smooth muscle cells.

PRINCIPAL FINDINGS

1. Caveolin-1 resides in the endoplasmic, not the cytoplasmic, leaflets of ER membranes
We used freeze-fracture immunocytochemistry to localize caveolin-1. Membranes always split into their two constituent leaflets during freeze-fracturing, revealing the P-face (cytoplasmic leaflet) or the E-face (endoplasmic leaflet), as the plane of cleavage runs preferentially between the tails of the phospholipids of the membrane bilayer. Whether immunolabeled caveolin-1 appears on the P-face or on the E-face is exquisitely revealed in the electron microscope after immunolabeling of evaporated metal replicas of the fractured surfaces with anticaveolin-1 and an appropriate colloidal gold conjugate.

It is unexpected that we found that caveolin-1 labeling is abundant on the E-faces (in the endoplasmic leaflets) but is completely absent on the P-faces (in the cytoplasmic leaflets) of all ER membranes (Fig. 1 ).

View larger version (134K): [in this window] [in a new window]   Figure 1. Caveolin-1 labeling on membranes of the ER in replicas of freeze-fractured smooth muscle cells. The E-faces (E) of ER cisternae are labeled, whereas the P-faces (P) are unlabeled. M, Mitochondrion. Original bar, 0.5 µm.

2. Caveolin-1 is targeted to the core of lipid droplets
Thin- and cryo-sectioned lipid droplets appeared relatively devoid of internal structure, but freeze-fracturing revealed numerous internal layers that were often arranged concentrically like the leaves of an onion, as well as amorphous-appearing areas in the droplet cores (Fig. 2 ). We found caveolin-1 labeling in notable amounts on some of the layers and at varying depths within the droplets in about half of the lipid droplets examined. Thus, caveolin-1 is present in the core of the lipid droplet.

View larger version (185K): [in this window] [in a new window]   Figure 2. Caveolin-1 labeling on lipid droplets. A) Concavely fractured droplet. Caveolin-1 is labeled on the apparently outermost layer and on a fragment of an inner one. B) Convexly fractured droplet. Layers at various depths, including the apparently outermost one, are strongly labeled. C) Convexly fractured droplet. In this droplet, the apparently outermost layer is unlabeled, but a less superficial one is. D) Cross-fractured droplet. Caveolin-1 is detected throughout the core, especially in association with nonconcentrically organized layers. Original bars, 0.25 µm.

Gold particles marking caveolin-1 were also located at the surfaces of lipid droplets. However, we could not ascertain unequivocally whether the label was on the cytoplasmic membrane leaflet enveloping the droplets or merely on subsurface layers of the core; fiducial identification of the outer surfaces of the droplets was confounded by the presence of the adjacent, internal layers.

CONCLUSIONS AND SIGNIFICANCE

Caveolin-1 molecules are presently assumed to be inserted into the cytoplasmic leaflets of ER membranes, and caveolin-1 is thought to move to lipid droplets only when the concentration of lipids in the ER is abnormally high. Our results showed that caveolin-1 resides in the endoplasmic leaflet of ER membranes but is absent in the cytoplasmic leaflet. Thus, caveolin-1 molecules are not directed to the lipid droplet surface by lateral diffusion in the cytoplasmic membrane leaflet, enveloping the lipid droplet as hitherto postulated (Fig. 3 A); caveolin-1 molecules are simply on the inappropriate side of the ER membrane for this mechanism to apply (Fig. 3B ). The presence of caveolin-1 in lipid droplet cores indicates that caveolin-1 does somehow gain access to the core of the lipid droplet. However, caveolin-1 molecules cannot enter the droplet core simply by diffusing in the endoplasmic membrane leaflet nor are they transferred to the droplet along with this leaflet, as the droplet is enveloped in the cytoplasmic leaflet, not the caveolin-1-containing endoplasmic leaflet. Caveolin-1 molecules must therefore first translocate out of the endoplasmic leaflet before moving into the droplet core. Whereas we entertain several possible mechanisms for caveolin-1 targeting to the lipid droplet, we conclude that it is the high inherent affinity of lipids for caveolin-1 that promotes initial extraction of caveolin-1 molecules from the endoplasmic leaflet of ER membranes and the subsequent partitioning of caveolin-1 into the lipid phase of the droplet. Accordingly, we put forward a model of lipid droplet formation in which caveolin-1 molecules are transferred by the lipids themselves into the droplet core during lipid droplet biogenesis (Fig. 3C ). Unresolved remains whether or not caveolin-1 finds its way into the envelope surrounding the lipid droplet, why only some lipid droplets contain caveolin-1, how the lipids in the droplet core become ordered to form layers, and why numerous immunofluorescence studies in the past have detected caveolin primarily at the surfaces of lipid droplets and not in the cores. Finally, we note that elevated lipid concentration in the ER is not a necessity for the incorporation of caveolin-1 into lipid droplets, as targeting caveolin-1 to lipid droplets obviously takes place in nonstimulated, "normally" cultured smooth muscle cells with presumably normal levels of lipids in the ER.

View larger version (43K): [in this window] [in a new window]   Figure 3. Schematic diagram showing how caveolin molecules are not targeted to lipid droplets (A, B) but how they may be (C). A) Current model of lipid droplet formation. Caveolin is assumed to reside in the cytoplasmic leaflets of ER membranes. The cytoplasmic leaflet becomes distended by lipids inundating between the two membrane leaflets. Caveolin molecules diffuse freely in the cytoplasmic leaflet and are transferred along with this leaflet to the surface of the nascent lipid droplet before the droplet buds off the ER. This model cannot apply, as caveolin-1 is resident in the endoplasmic leaflet of ER membranes. B) True distribution of caveolin-1 in the endoplasmic leaflet of ER membranes. Caveolin-1 cannot directly access the droplet by the above mechanism at all, as caveolin-1 is on the wrong side of the ER membrane. C) Updated model of lipid droplet formation. The high inherent affinity of lipids for caveolin causes caveolin molecules to be extracted out of the endoplasmic leaflet by inundating neutral lipids and to be transferred into the lipid core of the nascent lipid droplet before budding takes place. This model agrees with the actual distribution of caveolin in ER membranes and in lipid droplet cores revealed by freeze-fracture immunocytochemical labeling.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0782fje;

² This paper is dedicated to the memory of Kazushi Fujimoto in tribute to him and his valuable work.

This article has been cited by other articles:

				G. Murphy Jr., R. L. Rouse, W. W. Polk, W. G. Henk, S. A. Barker, M. J. Boudreaux, Z. E. Floyd, and A. L. Penn Combustion-Derived Hydrocarbons Localize to Lipid Droplets in Respiratory Cells Am. J. Respir. Cell Mol. Biol., May 1, 2008; 38(5): 532 - 540. [Abstract] [Full Text] [PDF]

				P. Pacheco, A. Vieira-de-Abreu, R. N. Gomes, G. Barbosa-Lima, L. B. Wermelinger, C. M. Maya-Monteiro, A. R. Silva, M. T. Bozza, H. C. Castro-Faria-Neto, C. Bandeira-Melo, et al. Monocyte Chemoattractant Protein-1/CC Chemokine Ligand 2 Controls Microtubule-Driven Biogenesis and Leukotriene B4-Synthesizing Function of Macrophage Lipid Bodies Elicited by Innate Immune Response J. Immunol., December 15, 2007; 179(12): 8500 - 8508. [Abstract] [Full Text] [PDF]

				M. Puhka, H. Vihinen, M. Joensuu, and E. Jokitalo Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells J. Cell Biol., December 3, 2007; 179(5): 895 - 909. [Abstract] [Full Text] [PDF]

				S. Y. Cho, E. S. Shin, P. J. Park, D. W. Shin, H. K. Chang, D. Kim, H. H. Lee, J. H. Lee, S. H. Kim, M. J. Song, et al. Identification of Mouse Prp19p as a Lipid Droplet-associated Protein and Its Possible Involvement in the Biogenesis of Lipid Droplets J. Biol. Chem., January 26, 2007; 282(4): 2456 - 2465. [Abstract] [Full Text] [PDF]

				H.-C. Wan, R. C. N. Melo, Z. Jin, A. M. Dvorak, and P. F. Weller Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies FASEB J, January 1, 2007; 21(1): 167 - 178. [Abstract] [Full Text] [PDF]

				T. FUJIMOTO and Y. OHSAKI Cytoplasmic Lipid Droplets: Rediscovery of an Old Structure as a Unique Platform Ann. N.Y. Acad. Sci., November 1, 2006; 1086(1): 104 - 115. [Abstract] [Full Text] [PDF]

				H. Robenek, O. Hofnagel, I. Buers, M. J. Robenek, D. Troyer, and N. J. Severs Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis J. Cell Sci., October 15, 2006; 119(20): 4215 - 4224. [Abstract] [Full Text] [PDF]

				D. T. Browman, M. E. Resek, L. D. Zajchowski, and S. M. Robbins Erlin-1 and erlin-2 are novel members of the prohibitin family of proteins that define lipid-raft-like domains of the ER J. Cell Sci., August 1, 2006; 119(15): 3149 - 3160. [Abstract] [Full Text] [PDF]

				H. Robenek, O. Hofnagel, I. Buers, S. Lorkowski, M. Schnoor, M. J. Robenek, H. Heid, D. Troyer, and N. J. Severs Butyrophilin controls milk fat globule secretion PNAS, July 5, 2006; 103(27): 10385 - 10390. [Abstract] [Full Text] [PDF]

				D. Binns, T. Januszewski, Y. Chen, J. Hill, V. S. Markin, Y. Zhao, C. Gilpin, K. D. Chapman, R. G.W. Anderson, and J. M. Goodman An intimate collaboration between peroxisomes and lipid bodies J. Cell Biol., June 5, 2006; 173(5): 719 - 731. [Abstract] [Full Text] [PDF]

				P. Kleinbongard, R. Schulz, T. Rassaf, T. Lauer, A. Dejam, T. Jax, I. Kumara, P. Gharini, S. Kabanova, B. Ozuyaman, et al. Red blood cells express a functional endothelial nitric oxide synthase Blood, April 1, 2006; 107(7): 2943 - 2951. [Abstract] [Full Text] [PDF]

				H. Robenek, M. J. Robenek, I. Buers, S. Lorkowski, O. Hofnagel, D. Troyer, and N. J. Severs Lipid Droplets Gain PAT Family Proteins by Interaction with Specialized Plasma Membrane Domains J. Biol. Chem., July 15, 2005; 280(28): 26330 - 26338. [Abstract] [Full Text] [PDF]

				S. Ozeki, J. Cheng, K. Tauchi-Sato, N. Hatano, H. Taniguchi, and T. Fujimoto Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane J. Cell Sci., June 15, 2005; 118(12): 2601 - 2611. [Abstract] [Full Text] [PDF]

				H. Robenek, M. J. Robenek, and D. Troyer PAT family proteins pervade lipid droplet cores J. Lipid Res., June 1, 2005; 46(6): 1331 - 1338. [Abstract] [Full Text] [PDF]

				H. Robenek, S. Lorkowski, M. Schnoor, and D. Troyer Spatial Integration of TIP47 and Adipophilin in Macrophage Lipid Bodies J. Biol. Chem., February 18, 2005; 280(7): 5789 - 5794. [Abstract] [Full Text] [PDF]

				Y. Imanishi, V. Gerke, and K. Palczewski Retinosomes: new insights into intracellular managing of hydrophobic substances in lipid bodies J. Cell Biol., August 16, 2004; 166(4): 447 - 453. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/7/866 03-0782fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ROBENEK, M. J. Articles by ROBENEK, H. Search for Related Content PubMed PubMed Citation Articles by ROBENEK, M. J. Articles by ROBENEK, H.