Identification, sequence and developmental expression of invertebrate flotillins from Drosophila melanogaster1
Introduction
Caveolae are small omega-shaped indentations of the plasma membrane that have been implicated in signal transduction and vesicular transport processes (Couet et al., 1997; Lisanti et al., 1994a, Lisanti et al., 1994b; Okamoto et al., 1998). Caveolae are most abundant in terminally differentiated cells, such as adipocytes, endothelial cells, smooth muscle cells, skeletal and cardiac myocytes and fibroblasts (Bretscher and Whytock, 1977; Fan et al., 1983; Forbes et al., 1979; Scherer et al., 1994, Scherer et al., 1996; Simionescu and Simionsecu, 1983).
Caveolin, a 21–24 kDa integral membrane protein, is a principal component of caveolae membranes in vivo (Glenney, 1989, Glenney, 1992; Glenney and Soppet, 1992; Kurzchalia et al., 1992; Rothberg et al., 1992). Caveolin-rich membrane domains have been purified by either detergent-based or detergent-free methods and they are enriched in a variety of lipid-modified signaling molecules such as hetero-trimeric G-proteins, Src-family tyrosine kinases, H-Ras and Rap GTPases, and ecNOS (Chang et al., 1994; Garcia-Cardena et al., 1996; Lisanti et al., 1994a, Lisanti et al., 1994b; Liu et al., 1996; Liu et al., 1997; Robbins et al., 1995; Sargiacomo et al., 1993; Shaul et al., 1996; Shenoy-Scaria et al., 1994; Smart et al., 1995; Song et al., 1996a, Song et al., 1996b). Many of these signaling molecules interact directly with caveolin, in a regulated manner (Li et al., 1995, Li et al., 1996; Song et al., 1996a, Song et al., 1996b).
However, caveolin is only the first member of a growing gene family of caveolin proteins; caveolin has been re-termed caveolin-1. Three different caveolin genes (Cav-1, Cav-2 and Cav-3) encoding four different subtypes of caveolin have been described thus far (Couet et al., 1997). There are two sub-types of caveolin-1 (Cav-1α and Cav-1β) that differ in their respective translation initiation sites (Scherer et al., 1995). The tissue distribution of caveolin-2 mRNA is extremely similar to that of caveolin-1 mRNA (Scherer et al., 1996, Scherer et al., 1997). In striking contrast, caveolin-3 mRNA and protein is expressed mainly in muscle tissue types (skeletal, cardiac and smooth) (Song et al., 1996a, Song et al., 1996b; Tang et al., 1996; Way and Parton, 1995).
Recently, we have identified another family of integral membrane proteins that may contribute to the structural organization of caveolae membranes (Bickel et al., 1997). Micro-sequence analysis of purified caveolin-rich membrane domains isolated from lung tissue revealed a novel approx. 45 kDa component of caveolae membranes, termed flotillin (Bickel et al., 1997). Molecular cloning of flotillin and analysis of the cDNA for this protein has provided new avenues by which to explore the structure and function of caveolae organelles. Interestingly, flotillin is a close homologue of ESA and together they define a new `flotillin family' of caveolae-associated integral membrane proteins (flotillin-1 and flotillin-2/ESA) (Bickel et al., 1997).
Although caveolae have been observed in neuronal plasma membranes by electron microscopy, no mRNA species for known caveolin gene family members have been detected in brain. In contrast, flotillin-1 mRNA and protein are expressed abundantly in brain tissue and flotillin-1 may serve as a marker protein for caveolae-like structures in neurons and the brain (Bickel et al., 1997).
The study of caveolae in vivo is currently hampered by a lack of genetic systems to dissect the requirements for expression of certain caveolar components. In this regard, we are interested in establishing invertebrate model systems to study the caveolin and flotillin gene families. As caveolins 1 and 2 have recently been identified in C. elegans (Tang et al., 1997), flotillin homologues should exist in invertebrates.
Here, we describe the identification, sequence and embryonic expression pattern of an invertebrate flotillin, FLODm, in Drosophila melanogaster. Localization of the FLODm message in D. melanogaster embryos reveals that expression of FLODm is confined primarily to the developing nervous system. This is consistent with our previous observation that mammalian flotillin-1 mRNA and protein is expressed abundantly in brain tissue (Bickel et al., 1997).
Section snippets
Materials
The D. melanogaster EST clone CK02126 was obtained from Dr John Pendelton (Dr Gerald M. Rubin Lab, HHMI, BDGP EST Project). Anti-myc IgG (9E10) were from Santa Cruz Biotechnologies. Anti-caveolin-1 IgG were from Santa Cruz Biotechnologies and Transduction Laboratories. Other reagents were from the following commercial suppliers: prestained protein markers, fetal bovine serum and other cell culture reagents (Life Technologies/ Gibco-BRL).
Recombinant expression of FLODm
An epitope-tagged form of the FLODm cDNA was subcloned
A flotillin homologue from Drosophila melanogaster: identification, cDNA sequence and protein sequence analysis
In order to identify an invertebrate flotillin gene, we searched existing databases of model invertebrate organisms using the protein sequence of mammalian flotillin-1 and flotillin-2 (also known as ESA). Using this approach, we identified an EST sequence potentially encoding a flotillin homologue in Drosophila melanogaster. However, it should be noted that the reported DNA sequence of this EST is limited, contains several errors and is out of frame in many instances.
To establish the correct
Acknowledgements
We thank members of the Lisanti, Stevens and Stein laboratories for helpful and insightful discussions. This work was supported by an NIH FIRST Award (GM-50443; to M.P.L.), a grant from the G. Harold and Leila Y. Mathers Charitable Foundation (to M.P.L.) and a Scholarship in the Medical Sciences from the Charles E. Culpeper Foundation (to M.P.L.). Work in L. Stevens' laboratory was supported by a grant from the NIH (GM 49853). Work in D. Stein's laboratory was also supported by the NIH.
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The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL Data Bank with accession Nos AF044734 and AF044916.