The second hydrophobic region of flotillin-1 is necessary for its plasma membrane localization, while the N-terminal sequences direct interactions with CAP and Fyn in 3T3-L1 adipocytes.
The study identifies distinct hydrophobic domains in flotillin-1 that direct its localization to the plasma membrane and lipid rafts, facilitating interactions with CAP, Fyn, and F-actin in adipocytes.
Flotillin-1 is a lipid raft-associated protein that has been implicated in various cellular processes. We examined the subcellular distribution of flotillin-1 in different cell types and found that localization is cell type-specific. Flotillin-1 relocates from a cytoplasmic compartment to the plasma membrane upon the differentiation of 3T3-L1 adipocytes. To delineate the structural determinants necessary for its localization, we generated a series of truncation mutants of flotillin-1. Wild type flotillin-1 has two putative hydrophobic domains and is localized to lipid raft microdomains at the plasma membrane. Flotillin-1 fragments lacking the N-terminal hydrophobic stretch are excluded from the lipid raft compartments but remain at the plasma membrane. On the other hand, mutants with the second hydrophobic region deleted fail to traffic to the plasma membrane but are instead found in intracellular granule-like structures. Flotillin-1 specifically interacts with the adaptor protein CAP, the Src family kinase Fyn, and cortical F-actin in lipid raft microdomains in adipocytes. Furthermore, CAP and Fyn associate with different regions in the N-terminal sequences of flotillin-1. These results furthered our understanding for how flotillin-1 can function as a molecular link between lipid rafts of the plasma membrane and a multimeric signaling complex at the actin cytoskeleton. Flotillin-1 is a lipid raft-associated protein that has been implicated in various cellular processes. We examined the subcellular distribution of flotillin-1 in different cell types and found that localization is cell type-specific. Flotillin-1 relocates from a cytoplasmic compartment to the plasma membrane upon the differentiation of 3T3-L1 adipocytes. To delineate the structural determinants necessary for its localization, we generated a series of truncation mutants of flotillin-1. Wild type flotillin-1 has two putative hydrophobic domains and is localized to lipid raft microdomains at the plasma membrane. Flotillin-1 fragments lacking the N-terminal hydrophobic stretch are excluded from the lipid raft compartments but remain at the plasma membrane. On the other hand, mutants with the second hydrophobic region deleted fail to traffic to the plasma membrane but are instead found in intracellular granule-like structures. Flotillin-1 specifically interacts with the adaptor protein CAP, the Src family kinase Fyn, and cortical F-actin in lipid raft microdomains in adipocytes. Furthermore, CAP and Fyn associate with different regions in the N-terminal sequences of flotillin-1. These results furthered our understanding for how flotillin-1 can function as a molecular link between lipid rafts of the plasma membrane and a multimeric signaling complex at the actin cytoskeleton. The plasma membrane of most cell types contains specialized subdomains that are highly enriched in cholesterol and sphingolipids, referred to as lipid raft microdomains (1Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 2Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8048) Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5133) Google Scholar, 4Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1719) Google Scholar). Lipid rafts are highly organized, dynamic structures connected to the cytoskeleton and are enriched in growth factor receptors, integrins, Src family kinases, glycosylphosphatidylinositol-linked proteins, and adaptor proteins (5Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar, 6Harris T.J. Siu C.H. 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The complexes were with protein for at and were with in proteins were and to proteins were with the and with fractions plasma membrane density and low density were from 3T3-L1 and a of and as and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells on were in for cells with for various were on with the cells in of and and were in a The cell were into various and the were in a protein of and to and were on in the with for cells were with for and with and for and were at in and the were on with were 3T3-L1 with the of were on The cells were to for and were for with the and on as were in for with the cells were for on in and The The insoluble in and as C.A. M. S. S. P.E. J.E. Nature. 2000; PubMed Scopus Google Scholar). of Flotillin-1 in the cellular in flotillin-1 has been we to and its intracellular localization in different cell To flotillin-1 at the with a and expressed in a series of cell via that flotillin-1 at membrane with a intracellular distribution in and cells, the protein localized at the plasma membrane with cytoplasmic in cells, flotillin-1 intracellular granule-like with a at the plasma membrane flotillin-1 can in plasma membrane and intracellular and the distribution between these two compartments to cell type-specific. The of Flotillin-1 in 3T3-L1 flotillin-1 is localized in we the distribution of flotillin-1 subcellular As shown in flotillin-1 in and fractions with a in fractions in 3T3-L1 and adipocytes. a of flotillin-1 in the to that in upon differentiation of differentiation the subcellular distribution of caveolin, a lipid raft protein enriched in the of To these we examined the localization of flotillin-1 in 3T3-L1 cells flotillin-1. As shown in flotillin-1 found in intracellular structures in with of these with the actin cytoskeleton. differentiation into flotillin-1 found to localized at the plasma where cortical F-actin also To the cellular compartments in that contain proteins of the and were As shown in the structures flotillin-1 in were but that flotillin-1 is in to cellular To differentiation also a of we expressed in and these cells to of growth in the that flotillin-1 localized in intracellular in and of flotillin-1 is also cell type-specific. To the flotillin-1 protein lipid raft localization in we 3T3-L1 and 3T3-L1 with low from and insoluble fractions were with different of flotillin-1 protein in the of the protein in the differentiation into flotillin-1 protein found in the Fyn, a Src family kinase that shown to interact with flotillin-1 in and cells, also a lipid raft As a caveolin found to localized to lipid rafts of the differentiation of the shown that lipid raft subdomains in the plasma membrane can as for actin cytoskeleton structures (3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5133) Google Scholar, 6Harris T.J. Siu C.H. BioEssays. 2002; 24: 996-1003Crossref PubMed Scopus (84) Google Scholar). To the role of the actin cytoskeleton in flotillin localization, we cells with the actin to the As shown in of actin cytoskeleton on the lipid raft localization of flotillin-1 caveolin in and adipocytes. The N-terminal for the Lipid of Flotillin-1 in the structural determinants for the localization of flotillin-1 to the lipid raft domains of we generated a series of truncation mutants of flotillin-1 at the with a We examined the of these on the flotillin-1 localization in 3T3-L1 As of flotillin-1 in a of the N-terminal that contain the hydrophobic stretch the the second hydrophobic stretch on of a that the N-terminal hydrophobic in a cytoplasmic localization with a of protein at the N-terminal that the second hydrophobic in intracellular The results that the second but the hydrophobic is for the of flotillin-1 to the plasma membrane of 3T3-L1 adipocytes. We examined the localization of and of flotillin-1 to the lipid raft microdomains of the We protein with various flotillin proteins in 3T3-L1 adipocytes. were with at low and the membrane were is that proteins in the are lipid the of flotillin-1 and caveolin from these structures cholesterol that is a for lipid raft localization of flotillin-1 As shown in the protein to the in a in the with its in lipid the flotillin to and in the membrane the with and from the membrane and were they also were these that the hydrophobic to the of flotillin-1 role in the protein to the lipid raft To the results from the cells were also to subcellular into and The distribution of various flotillin mutants between these two fractions examined As shown in although of the protein found in the of and of were localized in the same is with the results from as the that in the intracellular compartments as found to in the the hydrophobic is necessary for flotillin-1 to lipid raft microdomains in and intracellular membrane structures. that of is in flotillin-1 with the in cells Rea S. S. R. J.F. Parton R.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). To of also is for the localization of flotillin in we 3T3-L1 with type of flotillin-1 with As the type flotillin with the in cells and localized with in in intracellular that also were of to on the subcellular localization of flotillin and has been in cells, the of to a role in the lipid raft localization of flotillin-1 in adipocytes. of the as the of of localization of and at the cell of to that the second hydrophobic between and flotillin-1 to the plasma membrane. To delineate the for localization, we various N-terminal of flotillin to the and expressed protein in 3T3-L1 adipocytes. As flotillin and were localized to and intracellular generated of the second hydrophobic stretch to in intracellular of from the protein the same on the localization as and of region On the other hand, and that the second hydrophobic well with the from the protein of the hydrophobic from its these that the second but the hydrophobic of flotillin-1 is for the localization and for the of the protein from intracellular structures. CAP at the of 3T3-L1 is a adaptor protein highly expressed in and Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). to a protein at the in K. H. K. K. H. J. Biol. PubMed Scopus Google Scholar) and a lipid raft in C.A. M. S. S. P.E. J.E. Nature. 2000; PubMed Scopus Google Scholar), we to CAP associate with the cortical actin cytoskeleton at the of adipocytes. on 3T3-L1 a to to for F-actin the of the cells, that CAP to the cortical F-actin with intracellular the plasma membrane CAP found to well with F-actin at the cell and the structures. CAP at the and the of actin at the plasma membrane we also found that CAP of to and F-actin in in and S. M. M. L. H. and R. of Flotillin-1 with that flotillin-1 CAP to plasma membrane lipid rafts in C.A. M. S. S. P.E. J.E. Nature. 2000; PubMed Scopus Google Scholar). To the of CAP with were cell from 3T3-L1 adipocytes. CAP proteins were with the of As were and As shown in a of flotillin-1 with CAP of CAP were The same of and were to CAP a between CAP and flotillin-1 in of the results in isoforms of CAP We M. Mol. PubMed Google Scholar) that were isoforms of CAP protein expressed in adipose To the of CAP to flotillin-1 is we cells with and a of the CAP with As shown in specifically with but with the other and the same the of flotillin with CAP is of with Flotillin-1 and that flotillin-1 and actin to these are in To we and in well that with actin and at at the of the cells, where found to at the of the to well with in the We with in 3T3-L1 Both CAP proteins to in cells were with we that the of with in the of cells with to cortical F-actin from the as well as the membrane On the other hand, the of cortical actin on the localization of flotillin in the lipid These results that is with flotillin-1 in the lipid raft is on the of the cortical F-actin of and Fyn in to the region of flotillin-1 for to and with with the same as the of the that the hydrophobic from to a protein that to a that a of the protein in the various These that the hydrophobic of flotillin-1 is for its with shown that flotillin-1 and flotillin-2 are with the Src family kinase Fyn in fractions of that flotillin role in the formation of centers (10Stuermer C.A. 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Fyn in the lacking flotillin-1. that the of Fyn the Fyn kinase a complex with flotillin-1 of its kinase To the region in we Fyn and various truncation mutants of flotillin-1. that of the N-terminal the of the second hydrophobic Fyn that these regions are for the N-terminal to the of the second hydrophobic in flotillin Fyn These results that the region between the two hydrophobic domains in flotillin the of the protein to To Fyn the of to cells were also with and in the of were with that the of with type kinase Fyn of on the between Fyn and flotillin in a These results that the regions in flotillin-1 for and Fyn with these proteins of in a multimeric and of and -2 are ubiquitously expressed lipid raft proteins that been with cellular from Stuermer and D.M. Lommel S. Jung M. Ankerhold R. Petrausch B. Laessing U. Wiechers M.F. Plattner H. Stuermer C.A. J. Neurobiol. 1998; 37: 502-523Crossref PubMed Scopus (202) Google Scholar, 10Stuermer C.A. Lang D.M. Kirsch F. Wiechers M. 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Liu et al. (Wed,) reported a other. Flotillin-1 truncation mutants vs. Wild type flotillin-1 was evaluated on Subcellular localization and protein interactions. The second hydrophobic region of flotillin-1 is necessary for its plasma membrane localization, while the N-terminal sequences direct interactions with CAP and Fyn in 3T3-L1 adipocytes.