Distinct targeting pathways for the membrane insertion of tail-anchored (TA) proteins

Tail-anchored (TA) proteins are characterized by a C-terminal transmembrane region that mediates posttranslational insertion into the membrane of the endoplasmic reticulum. We have investigated the requirements for membrane insertion of three TA proteins, RAMP4, Sec61β and cytochrome b5. We show here that newly synthesized RAMP4 and Sec61β can accumulate in a cytosolic, soluble complex with the ATPase Asna-1/TRC40 before insertion into ER-derived membranes. Membrane insertion of these TA proteins is stimulated by ATP, sensitive to redox conditions and blocked by alkylation of SH groups by N-ethylmaleimide (NEM). In contrast, membrane insertion of cytochrome b5 is not found to be mediated by Asna-1, not stimulated by ATP and not affected by NEM or an oxidative environment. Asna-1 mediated pathway of membrane insertion of RAMP4 and Sec61β may relate to functions of these proteins in the ER stress response

Transsynaptic Teneurin Signaling in Neuromuscular Synapse Organization and Target Choice. Nature. 2012; (this issue

Synapse assembly requires trans-synaptic signals between the preand postsynapse 1 , but our understanding of the essential organizational molecules involved in this process remains incomplete 2 . Teneurin proteins are conserved, epidermal growth factor (EGF)-repeat-containing transmembrane proteins with large extracellular domains Vertebrate teneurins are enriched in the developing brain Both Ten-m and Ten-a were enriched at the larval NMJ ( The localization of Ten-a and Ten-m suggested their trans-synaptic interaction. To examine this, we co-expressed Myc-tagged Ten-a in nerves using the Q system 14 and haemagglutinin (HA)-tagged Ten-m in muscles using GAL4. Muscle Ten-m was able to coimmunoprecipitate nerve Ten-a from larval synaptosomes To determine Teneurin function at the NMJ, we examined the ten-a null allele and larvae with neuron or muscle RNAi of ten-a and/or ten-m. Following such perturbations, bouton number and size were altered: the quantity was reduced by 55

Transforming Growth Factor Beta 1 and Vascular Endothelial Growth Factor Levels in the Pathogenesis of Periodontal Disease

Periodontal disease is characterized by inflammation and bone loss. The balance between inflammatory mediators and their counter-regulatory molecules may be fundamental for determining the outcome of immune pathology of periodontal disease. Cytokines play crucial roles in the maintenance of tissue homeostasis, a process which requires a delicate balance between anabolic and catabolic activities. In particular, two families of growth factors-such as transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) are thought to play important roles in modulating the proliferation and/or migration of structural cells involved in inflammation and regulation of immune responses. The aim of this work was to analyze gingival samples and periodontal tissue specimens collected from thirty-eight patients with chronic periodontal disease and from forty healthy individuals, in order to detect the expression and distribution of TGF-β1 and VEGF between the two groups. TGF-β1 and VEGF expression levels were detected using immunohistochemical analysis and computer-assisted morphometric analysis. The findings presented here suggest that biomarker such as TGF-β1 and VEGF have an important regulating role in the orchestration of the immune response, which in turn influence the outcome of disease establishment and evolution

Structural insights into tail-anchored protein binding and membrane insertion by Get3

Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and α-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the α-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the α-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-Mg2+- and ADP-Mg2+-bound crystal structures representing the pre- and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP·AlF4−-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361–366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3

EBV protein BNLF2a exploits host tail-anchored protein integration machinery to inhibit TAP

EBV, the prototypic human γ(1)-herpesvirus, persists for life in infected individuals, despite the presence of vigorous antiviral immunity. CTLs play an important role in the protection against viral infections, which they detect through recognition of virus-encoded peptides presented in the context of HLA class I molecules at the cell surface. The viral peptides are generated in the cytosol and are transported into the endoplasmic reticulum (ER) by TAP. The EBV-encoded lytic-phase protein BNLF2a acts as a powerful inhibitor of TAP. Consequently, loading of antigenic peptides onto HLA class I molecules is hampered, and recognition of BNLF2a-expressing cells by cytotoxic T cells is avoided. In this study, we characterize BNLF2a as a tail-anchored (TA) protein and elucidate its mode of action. Its hydrophilic N-terminal domain is located in the cytosol, whereas its hydrophobic C-terminal domain is inserted into membranes posttranslationally. TAP has no role in membrane insertion of BNLF2a. Instead, Asna1 (also named TRC40), a cellular protein involved in posttranslational membrane insertion of TA proteins, is responsible for integration of BNLF2a into the ER membrane. Asna1 is thereby required for efficient BNLF2a-mediated HLA class I downregulation. To optimally accomplish immune evasion, BNLF2a is composed of two specialized domains: its C-terminal tail anchor ensures membrane integration and ER retention, whereas its cytosolic N terminus accomplishes inhibition of TAP function. These results illustrate how EBV exploits a cellular pathway for TA protein biogenesis to achieve immune evasion, and they highlight the exquisite adaptation of this virus to its host