Search

Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can post-translationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533-2542). In the present study, we investigated the limits of this unassisted translocation and report that surprisingly long (85 residues) domains of different sequence and charge placed downstream of b5's TMD can posttranslationally translocate into mammalian microsomes and liposomes at nanomolar nucleotide concentrations. Furthermore, integration of these constructs occurred in vivo in translocon-defective yeast strains. Unassisted translocation was not unique to b5 but was also observed for another TA protein (protein tyrosine phosphatase 1B) whose TMD, like the one of b5, is only moderately hydrophobic. In contrast, more hydrophobic TMDs, like synaptobrevin's, were incapable of supporting unassisted integration, possibly because of their tendency to aggregate in aqueous solution. Our data resolve long-standing discrepancies on TA protein insertion and are relevant to membrane evolution, biogenesis, and physiology.

We have recently shown that oxytocin inhibits cell growth when the vast majority of oxytocin receptors (OTRs) are excluded from detergent-resistant membranes (DRNs); the biochemical counterpart of lipid rafts), but has a strong mitogenic effect when the receptors are targeted to these plasma membrane domains upon fusion with caveolin-2, a resident raft protein. The aim of this study was to investigate whether the manipulation of total cell cholesterol can influence OTR localization and signaling. Our data indicate that cholesterol depletion in HEK-293 cells does not affect the signaling events mediated by the OTRs located outside DRMs. When treated 2 nM methyl-[beta]-cyclodextrin (M[beta]CD), the receptors remained outside and continue to inhibit cell growth. On the contrary, (M[beta]CD) treatment of cells expressing receptors fused to caveolin-2 led to their redistribution outside DRMs, and converted the receptor-mediated proliferative effect into cell growth inhibition. These data indicate that 1) once released from DRMs, the receptors fused to caveolin-2 signal exactly as wild-type OTRs and 2) their DRM location is responsible for the specific OTR signaling leading to cell proliferation. Finally, we evaluated whether cholesterol loading could force the OTRs into lipid rafts and change their signaling, but, after cell treatment with an MI3CD/cholesterol complex, receptor stimulation continued to lead to cell growth inhibition, thus indicating that increasing cell cholesterol levels is not sufficient per se to affect OTR signaling. lipid rafts; proliferation; neurohypophyseal hormones

VAPB (vesicle-associated membrane protein-associated protein B) is an endoplasmic reticulum (ER)-resident tail-anchored adaptor protein involved in lipid transport. A dominantly inherited mutant, P56S-VAPB, causes a familial form of amyotrophic lateral sclerosis (ALS) and forms poorly characterized inclusion bodies in cultured cells. To provide a cell biological basis for the understanding of mutant VAPB pathogenicity, we investigated its biogenesis and the inclusions that it generates. Translocation assays in cell-free systems and in cultured mammalian cells were used to investigate P56S-VAPB membrane insertion, and the inclusions were characterized by confocal imaging and electron microscopy. We found that mutant VAPB inserts post-translationally into ER membranes in a manner indistinguishable from the wild-type protein but that it rapidly clusters to form inclusions that remain continuous with the rest of the ER. Inclusions were induced by the mutant also when it was expressed at levels comparable to the endogenous wild-type protein. Ultrastructural analysis revealed that the inclusions represent a novel form of organized smooth ER (OSER) consisting in a limited number of parallel cisternae (usually 2 or 3) interleaved by a ~30 nm-thick electron-dense cytosolic layer. Our results demonstrate that the ALS-linked VAPB mutant causes dramatic ER restructuring that may underlie its pathogenicity in motoneurons. [ABSTRACT FROM AUTHOR]