VIP21-caveolin is a membrane protein, proposed to be a component of the striated coat covering the cytoplasmic surface of caveolae. To investigate the biochemical composition of the caveolar coat, we used our previous observation that VIP21-caveolin is present in large complexes and insoluble in the detergents CHAPS or Triton X-114. The mild treatment of these insoluble structures with sodium dodecyl sulfate leads to the detection of high molecular mass complexes of approximately 200, 400, and 600 kDa. The 400-kDa complex purified to homogeneity from dog lung is shown to consist exclusive of the two isoforms of VIP21-caveolin. Pulse-chase experiments indicate that the oligomers form early after the protein is synthesized in the endoplasmic reticulum (ER). VIP21-caveolin does indeed insert into the ER membrane through the classical translocation machinery. Its hydrophobic domain adopts an unusual loop configuration exposing the N-and C-flanking regions to the cytoplasm. Similar high molecular mass complexes can be produced from the in vitro-synthesized VIP21-caveolin. The complex formation occurs only if VIP21-caveolin isoforms are properly inserted into the membrane; formation is cytosol-dependent and does not involve a vesicle fusion step. We propose that high molecular mass oligomers of VIP21-caveolin represent the basic units forming the caveolar coat. They are formed in the ER and later, between the ER and the plasma membrane, these oligomers could associate into larger detergent-insoluble structures.

Cholesterol transport is an essential process in all multicellular organisms. In this study we applied two recently developed approaches to investigate the distribution and molecular mechanisms of cholesterol transport in Caenorhabditis elegans. The distribution of cholesterol in living worms was studied by imaging its fluorescent analog, dehydroergosterol, which we applied to the animals by feeding. Dehydroergosterol accumulates primarily in the pharynx, nerve ring, excretory gland cell, and gut of L1-L3 larvae. Later, the bulk of dehydroergosterol accumulates in oocytes and spermatozoa. Males display exceptionally strong labeling of spermatids, which suggests a possible role for cholesterol in sperm development. In a complementary approach, we used a photoactivatable cholesterol analog to identify cholesterol-binding proteins in C. elegans. Three major and several minor proteins were found specifically cross-linked to photocholesterol after UV irradiation. The major proteins were identified as vitellogenins. rme-2 mutants, which lack the vitellogenin receptor, fail to accumulate dehydroergosterol in oocytes and embryos and instead accumulate dehydroergosterol in the body cavity along with vitellogenin. Thus, uptake of cholesterol by C. elegans oocytes occurs via an endocytotic pathway involving yolk proteins. The pathway is a likely evolutionary ancestor of mammalian cholesterol transport.

The establishment of polarity in the embryo is fundamental for the correct development of an organism [1]. The first cleavage of the Caenorhabditis elegans embryo is asymmetric with certain cytoplasmic components being distributed unequally between the daughter cells [2-4]. Using a genetic screen, Kemphues and co-workers have identified six par genes (partition-defective) [5,6], which are involved in the process of asymmetric division. One of these genes encodes a highly conserved protein, PAR-1, which is a serine/threonine kinase that localizes asymmetrically to the posterior part of the zygote and to those blastocysts that give rise to the germ line [7-9]. We reasoned that the mammalian homologue of PAR-1 (mPAR-1) might be involved in the process of polarization of epithelial cells, which consist of apical and basolateral membrane domains. We found that mPAR-1 was expressed in a wide variety of epithelial tissues and cell lines and was associated with the cellular cortex. In polarized epithelial cells, mPAR-1 was asymmetrically localized to the lateral domain. A fusion protein lacking the kinase domain had the same localization as the full-length protein but its prolonged expression acted in a dominant-negative fashion: lateral adhesion of the transfected cells to neighbouring cells was diminished, resulting in the former cells being 'squeezed out' from the monolayer. Moreover, the polarity of these cells was disturbed resulting in mislocalization of E-cadherin. Thus, in the C. elegans embryo and in epithelial cells, polarity appears to be governed by similar mechanisms.

mall invaginations called caveolae are present on the surface of many cells and are a form of glycosphingolipid-and cholesterol-enriched microdomains or rafts in the plasma membrane 1,2 . The main component of the caveolar coat is caveolin-1 (refs 3,4), a membrane protein of relative molecular mass (M r ) 21,000 (21K), which binds cholesterol 5 and can form highmolecular-mass homo-oligomers resistant to sodium dodecyl sulphate (SDS) 6,7 . Caveolin-1 has been implicated in signal transduction, but its function remains unclear; whereas overactivation of the p42/44 MAP-kinase cascade was observed after downregulation of caveolin-1 (ref. 8), integrin-mediated activation of the Ras/ extracellular-signal-regulated kinase (ERK) pathway 9 or conversion of prostate cancer cells to an androgen-insensitive phenotype 10 required expression of caveolin-1. To resolve the function of caveolin-1 in intact animals, we analysed caveolin-1 and glycosphingolipid/cholesterol-enriched rafts during the development of the nematode Caenorhabditis elegans. We show that C. elegans caveolin-1 (CAV-1) is expressed in the adult germ line and during embryonic development, and that CAV-1 is essential for Ras/MAP-kinase-dependent progression through the meiotic cell cycle. The function of CAV-1 is dependent on its association with cholesterol-rich membrane microdomains, providing a link between the membrane composition of germ cells and meiotic progression. Our results demonstrate that caveolin-1 and cholesterol-rich microdomains have an essential role in signal transduction in vivo and suggest a model for meiotic progression in the C. elegans germ line.C. elegans has two caveolin-like genes, encoding CAV-1 and CAV-2 (ref. 11, and J.S. and T.V.K., unpublished). CAV-1 is about 67% similar and 37% identical to the mammalian caveolins-1 and -3 respectively over their entire length, whereas CAV-2 is a more distantly related member of the caveolin family 11 . Polyclonal antibodies produced against the carboxy-terminal peptide of CAV-1 recognize a single band of about 31K in western blots of C. elegans lysates (Fig. 1a). CAV-1 is expressed at low levels in adults (Fig. 1b), where it is found in germ cells (Fig. 1d). The protein is strongly expressed in most, if not all, cells throughout embryonic development (Fig. 1b,e,f). In late embryos and L1 larvae, cav-1 expression becomes restricted to the nervous system and diminishes during larval development (data not shown).The function of CAV-1 in C. elegans was studied by RNA interference (RNAi) 12 , a powerful method of obtaining phenocopies of C. elegans mutants by RNA injection into hermaphrodites. Treatment with RNAi for cav-1, after which CAV-1 is no longer detectable (data not shown), results in a burst of egg laying by the injected hermaphrodite. One hundred and twelve eggs (s.d. = 3) were laid by injected hermaphrodites (n = 30) within 36 h after injection of late L4 larvae, compared with 46 eggs laid (s.d. = 2) by control animals (n = 30). Behaviour, morphology and lifespan of injected hermaphrodi...

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor elF-2 in the quaternary initiation complex [elF-2 x GMPPCP x [aH]Met-tRNAf x 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and $24 were found to be unable to bind the ternary initiation complex [elF-2 x GMPPCP x pH]Met-tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins $2, $3 and S 17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S 13/ 16, S!9 and $24 are concluded to be involved in elF-2 binding to the 40 S subunit.40 S ribosomal subunit; elF-2 binding; Inhibitory antibody; Ribosomal protein