Caveolin-1 and -2 in the exocytic pathway of MDCK cells.

We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.

Genomic organization and transcriptional analysis of the human genes coding for caveolin-1 and caveolin-2.

Caveolin-1 and caveolin-2 are related proteins involved in the biogenesis of caveolae. The corresponding genes in humans (CAV and CAV2, respectively), have been mapped to a common locus in chromosome 7q31.1, and are possible candidates for the tumor suppressor gene postulated in this region. Here, we show that CAV and CAV2 are independent transcriptional units lying in the same orientation, with CAV2 centromeric and about 17kb upstream to CAV. The two genes have similar tissue expression patterns. Alternative termination/polyadenylation generates two CAV2 mRNAs. Multiple transcriptional start sites spanning 35bp upstream from the CAV2 ATG are detected by 5' RACE, consistent with a TATA-less promoter predicted by sequence analysis. The CAV2 promoter region contains two SRE-like boxes resembling those described in the CAV promoter and proposed to link transcription to intracellular cholesterol levels. However, exogenous sterols had only minor effects on CAV and CAV2 RNA levels in HeLa cells, suggesting that SREBPs are not sufficient to regulate caveolin transcription.

A photo-reactive derivative of ganglioside GM1 specifically cross-links VIP21-caveolin on the cell surface.

Previous studies have shown that sphingolipids may be enriched in caveolae, plasmalemmal invaginations implicated in endocytosis and signal transduction. We synthesised a radiolabeled derivative of ganglioside GM1 bearing a photo-reactive cross-linker at the end of its acyl chain. After insertion in the plasma membrane of cultured A431 or MDCK cells and photoactivation, the main protein cross-linked by the GM1 derivative was VIP21-caveolin, an essential structural component of caveolae. This result shows close proximity between GM1 molecules and VIP21-caveolin in the caveolar membrane and strongly implicates sphingolipid segregation in the biogenesis of caveolae.

Modulating secretion of antibodies.

Cells have means to ensure that only properly folded and assembled molecules are transported to their final destination, a phenomenon referred to as "quality control" of protein synthesis. Thus, plasma cells secrete only the polymeric form of IgM, retaining and degrading intracellularly assembly intermediates. Due to the failure to polymerize secretory IgM, B lymphocytes do not secrete IgM, while they express the membrane form of IgM on their surface. The selective retention of IgM assembly intermediates is due to disulphide interchange reactions which involve the C terminal cysteine of secretory microseconds chains and unknown protein(s) of the endoplasmic reticulum (ER). Assembly inhibits these reactions, as does addition of reducing agents. In the latter condition, assembly intermediates, otherwise retained and degraded in the ER, are transported to the Golgi, glycosylated and secreted. The developmental control of immunoglobulin secretion is discussed in the more general context of "quality control" of newly synthesized protein within the exocytic compartment.

De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.

Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae. Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein. Thy1, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.

Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events.

Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, mu and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of mu and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all mu chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of mu chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates.

The efficiency of cysteine-mediated intracellular retention determines the differential fate of secretory IgA and IgM in B and plasma cells.

Previous studies on IgM secretion demonstrated a role for the mu chain C-terminal cysteine (Cys575) in preventing the transport of unpolymerized subunits along the secretory pathway. The sequence homology between the C-terminal tailpieces of mu and alpha heavy chains prompted us to investigate the role of cysteine-mediated, retention in the control of IgA secretion during B cell development. Similar to IgM, IgA are not secreted by B lymphocytes: the retention mechanism can be reversed by the reducing agent 2-mercaptoethanol, suggesting that disulfide interchange reactions are involved in the quality control of both IgM and IgA. Yet, alpha 2L2 subunits, but not mu2L2, are secreted constitutively by plasma cells. We demonstrate that the differential retention of IgM and IgA subunits by myeloma transfectants is mainly due to the presence of an acidic residue upstream the alpha chain C-terminal cysteine. The regulation of polymeric Ig secretion during B cell development provides an example of how thiol-mediated quality control can be modulated according to the aminoacidic context surrounding the critical cysteine and to the cell type.

Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae.

Antibody binding to glycolipids and glycophosphatidylinositol (GPI)-anchored proteins of lymphocytes can trigger activation of specific signal transduction pathways. The finding that GPI-anchored proteins are present in detergent-insoluble complexes with several tyrosine kinases of the Src family suggested that these complexes may represent membrane microdomains involved in the transduction of signals to the cell interior. Recent work has suggested a link between detergent-insoluble microdomains and plasma membrane invaginations termed caveolae. Here we show that lymphocytes lack plasma membrane domains with the characteristic features of caveolae. Furthermore, VIP21-caveolin was not detectable in four different lymphocyte cell lines at the protein or mRNA level. In addition to the lack of caveolar domains, capping experiments suggested that the bulk of the GPI-anchored protein Thy1 and the glycosphingolipid GM1 were not stably associated in the lymphocyte plasma membrane. Despite this, Thy1 and GM1 were present in detergent-insoluble complexes. We conclude that detergent insolubility does not correlate with the presence of caveolae or of VIP21-caveolin and that caveolae, as defined by a number of different markers, are not involved in signal transduction in lymphocytes.

Exposed thiols confer localization in the endoplasmic reticulum by retention rather than retrieval.

The cysteine present in the Ig micro chain tailpiece (microtp) prevents the secretion of unpolymerized IgM intermediates and causes their accumulation in the endoplasmic reticulum (ER). In principle, this can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of the two mechanisms underlies the cysteine-dependent ER localization, we analyze here the post-translational modifications of suitably engineered cathepsin D (CD) molecules. The glycans of this protease are phosphorylated by post-ER phosphotransferases and further modified in the trans-Golgi to generate a mannose 6-phosphate lysosome targeting signal. Only trace amounts of the mutp-tagged CD (CDM&mutpCys) are phosphorylated, unless retention is reversed by exogenous reducing agents or the critical cysteine mutated (CDMmutpSer). In contrast, a KDEL-tagged CD, that is retrieved from the Golgi into the ER, acquires phosphates, though mainly resistant to alkaline phosphatase. Similarly to CDMmutpSer, the few CDMmutpCys molecules that escape retention and acquire phosphates in the cis-Golgi are transported beyond the KDEL retrieval compartment, as indicated by their sensitivity to alkaline phosphatase. These results demonstrate that the thiol-dependent ER localization arises primarily from true retention, without recycling through the Golgi.

Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation.

Plasma cells secrete IgM only in the polymeric form: the C-terminal cysteine of the mu heavy chain (Cys575) is responsible for both intracellular retention and assembly of IgM subunits. Polymerization is not quantitative, and part of IgM is degraded intracellularly. Neither chloroquine nor brefeldin A (BFA) inhibits degradation, suggesting that this process occurs in a pre-Golgi compartment. Degradation of IgM assembly intermediates requires Cys575: the monomeric IgMala575 mutant is stable also when endoplasmic reticulum (ER) to Golgi transport is blocked by BFA. Addition of the 20 C-terminal residues of mu to the lysosomal protease cathepsin D is sufficient to induce pre-Golgi retention and degradation of the chimeric protein: the small amounts of molecules which exit from the ER are mostly covalent dimers. By contrast, when retained by the KDEL sequence, cathepsin D is stable in the ER, indicating that retention is not sufficient to cause degradation. Replacing the C-terminal cysteine with serine restores transport through the Golgi. As all chimeric cathepsin D constructs display comparable protease activity in vitro, their different fates are not determined by gross alterations in folding. Thus, also out of its normal context, the mu chain Cys575 plays a crucial role in quality control, mediating assembly, retention and degradation.