1,720,990 research outputs found
Unassisted translocation of large polar domains across phospholipid bilayers
No full textAlthough 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 posttranslationally 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
How tails guide tail-anchored proteins to their destinations
No full textA large group of diverse, functionally important, and differently localized transmembrane proteins shares a particular membrane topology, consisting of a cytosolic N-terminal region, followed by a transmembrane domain close to the C-terminus. Because of their structure, these C-tail-anchored (TA) proteins must insert into all their target membranes by post-translational pathways. Recent work, based on the development of stringent and sensitive biochemical assays, has demonstrated that novel unexplored mechanisms underlie these post-translational targeting and membrane insertion pathways. Unravelling these pathways will shed light on the biosynthesis and regulation of an important group of membrane proteins and is likely to lead to new concepts in the field of membrane biogenesis
Endoplasmic reticulum architecture: structures in flux
No full textThe endoplasmic reticulum (ER) is a dynamic pleiomorphic organelle containing continuous but distinct subdomains. The diversity of ER structures parallels its many functions, including secretory protein biogenesis, lipid synthesis, drug metabolism and Ca2+ signaling. Recent studies are revealing how elaborate ER structures arise in response to subtle changes in protein levels, dynamics, and interactions as well as in response to alterations in cytosolic ion concentrations. Subdomain formation appears to be governed by principles of self-organization. Once formed, ER subdomains remain malleable and can be rapidly transformed into alternative structures in response to altered conditions. The mechanisms that modulate ER structure are likely to be important for the generation of the characteristic shapes of other organelles
Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa
No full textPlasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light p eak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane
Mechanism of precise intracellular targeting of spontaneously inserting tail-anchored proteins
No full textTail-anchored (TA) proteins are membrane proteins that are targeted to their destination by unique post-translational pathways. Cytochrome b5 is a spontaneously inserted TA protein, of which two forms are known: one targeting the ER (b5-ER) and other targeting the MOM (b5-RR). Microinjection of the recombinant proteins into cells results in precise targeting of each of the two proteins, indicating that the targeting information is present in the protein. Using digitonin semi-permeabilized cells and in the presence of rabbit reticulocyte lysate (RRL), we have obtained faithful targeting for both forms of cytochrome b5 that approaches the in cellula situation. In contrast, in the absence of cytosol both forms target the mitochondria. Attempting to find the chaperone responsible for the ER targeting, we observed that both TCR40 and Snd2 pathways play only a modest role, suggesting the contribution of redundant pathways to the biogenesis of b5. Recently, we found that Eyerestatin I, a p97 inhibitor, strongly inhibits the glycosylation of b5-ER. The effect seems to be specific for spontaneously inserted tail-anchored proteins, but the exact mechanism is still under investigation
Intracellular targeting of tail-anchored proteins
No full textTail-anchored (TA) proteins are integral membrane proteins that carry out important and diverse functions and that are targeted to their destination by unique post-translational pathways. Although the main pathway for targeting to the endoplasmic reticulum (ER) of TA proteins is represented by the TRC40/Get3 pathway, many TA proteins, i.e., those targeted in vivo to the mitochondrial outer membrane (MOM) and some ER targeted ones, can insert in vitro into pure phospholipid bilayers without assistance from any chaperone. The mechanism of precise in vivo targeting of these TA proteins is unclear. Cytochrome b5 is a spontaneously inserted TA protein, of which two forms are known, targeting the ER (b5-ER) or the MOM (b5-RR). The recombinant proteins microinjected into cultured cells are faithfully targeted, indicating that the targeting information is present in the protein and not in the mRNA. Using digitonin semi-permeabilized cells and in the presence of rabbit reticulocyte lysate as the source of cytosol, we have obtained faithful targeting for both forms of cytochrome b5 that approaches the in cellula situation. In contrast, in the absence of cytosol both forms target the mitochondria. We tested also the effects of energy depletion of the RRL and we observed an effect on b5-ER, confirmed by the reduction of ER localization. Thus, energy-dependent chaperones are required for b5-ER’s avoidance of the MOM and its specific targeting to the ER. Taking this into consideration, we investigated the role of the principal TA protein target TRC40 system on b5-ER targeting, by using two different approaches in our system: the coiled-coil domain of WRB (ER membrane receptor of TRC40), which act as a decoy receptor, and WRB silencing. These experiments demonstrated that TCR40 plays only a modest role in the post-translational delivery of b5-ER to the ER membrane, suggesting the contribution of redundant pathways to b5 biogenesis
Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells
No full textThe lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy
Transmembrane domain-dependent partitioning of membrane proteins within the endoplasmic reticulum
No full textThe length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein-protein and protein-lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail-anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER-Golgi boundary by simple receptor-independent mechanisms based on partitioning
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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