Lipid Raft Proteomics: More than Just Detergent-Resistant Membranes

2007 ◽  
pp. 35-47 ◽  
Author(s):  
Leonard J. Foster ◽  
Queenie W. T. Chan
Keyword(s):  
Lipid Raft ◽  
Detergent Resistant Membranes

scholarly journals Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft

2004 ◽  
Vol 78 (7) ◽  
pp. 3480-3488 ◽  
Author(s):  
Lu Gao ◽  
Hideki Aizaki ◽  
Jian-Wen He ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Raft ◽  
Critical Role ◽  
Rna Replication ◽  
Dominant Negative ◽  
Host Protein ◽  
Hcv Rna ◽  
Replication Complex ◽  
Detergent Resistant Membranes

ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.

scholarly journals Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

2010 ◽  
Vol 430 (3) ◽  
pp. 519-529 ◽  
Author(s):  
Karin Klappe ◽  
Anne-Jan Dijkhuis ◽  
Ina Hummel ◽  
Annie van Dam ◽  
Pavlina T. Ivanova ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Rafts ◽  
Cell Lines ◽  
Lipid Raft ◽  
Related Protein ◽  
Gradient Distribution ◽  
Caveolin 1 ◽  
Free Lipid ◽  
Detergent Resistant Membranes ◽  
Multidrug Resistance Related Protein

We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C16 species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.

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