Recognition and Chaperoning by Pex19, Followed by Trafficking and Membrane Insertion of the Peroxisome Proliferation Protein, Pex11

Pex11, an abundant peroxisomal membrane protein (PMP), is required for division of peroxisomes and is robustly imported to peroxisomal membranes. We present a comprehensive analysis of how the Pichia pastoris Pex11 is recognized and chaperoned by Pex19, targeted to peroxisome membranes and inserted therein. We demonstrate that Pex11 contains one Pex19-binding site (Pex19-BS) that is required for Pex11 insertion into peroxisomal membranes by Pex19, but is non-essential for peroxisomal trafficking. We provide extensive mutational analyses regarding the recognition of Pex19-BS in Pex11 by Pex19. Pex11 also has a second, Pex19-independent membrane peroxisome-targeting signal (mPTS) that is preserved among Pex11-family proteins and anchors the human HsPex11γ to the outer leaflet of the peroxisomal membrane. Thus, unlike most PMPs, Pex11 can use two mechanisms of transport to peroxisomes, where only one of them depends on its direct interaction with Pex19, but the other does not. However, Pex19 is necessary for membrane insertion of Pex11. We show that Pex11 can self-interact, using both homo- and/or heterotypic interactions involving its N-terminal helical domains. We demonstrate that Pex19 acts as a chaperone by interacting with the Pex19-BS in Pex11, thereby protecting Pex11 from spontaneous oligomerization that would otherwise cause its aggregation and subsequent degradation.

Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors

Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.

A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~45 residues away from the peptidyl transferase center. However, we recently uncovered instances where the first contact is delayed by up to ~10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the BtuC protein: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion.

Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages

Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.

Rapid Shear Stress-dependent ENaC Membrane Insertion is Mediated by the Endothelial Glycocalyx and the Mineralocorticoid Receptor

The contribution of the shear-stress sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR-/- mice. Flow- and acetylcholine-mediated vasodilation were measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro and ex vivo endothelial cells derived from C57BL/6 and ApoE/LDLR-/- vessel. As expected, in vivo in C57BL/6 vessels ENaC- and MR-inhibtion blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR-/- vessels this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction.

Discovery of a conserved rule behind the assembly of β-barrel membrane proteins

Gram-negative bacteria, mitochondria and chloroplasts contain β-barrel outer membrane proteins (OMPs). Most OMPs have a “β-signal” imprinted in the final β-strand. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex recognize the β-signal for the folding and membrane insertion of the OMP. Here, we identified the “-5 signal”, a novel signal existing in the fifth β-strand from the C-terminus (-5 strand) responsible for the insertion step of the assembly process. We further identified the receptor for the -5 signal as BamD. BamD can recognize both β-signal and -5 signal, marshalling the OMP for assembly. There is sequence similarity in of both signals observed also in mitochondrial OMPs. Therefore, we propose the “-5 rule” repeating a similar sequence in the -5 and last strand, as a conserved feature of the OMP assembly process in bacteria and eukaryotes.

α-Actinin 4 Links Vasopressin Short-Term and Long-Term Regulation of Aquaporin-2 in Kidney Collecting Duct Cells

Water permeability of the kidney collecting ducts is regulated by the peptide hormone vasopressin. Between minutes and hours (short-term), vasopressin induces trafficking of the water channel protein aquaporin-2 to the apical plasma membrane of the collecting duct principal cells to increase water permeability. Between hours and days (long-term), vasopressin induces aquaporin-2 gene expression. Here, we investigated the mechanisms that bridge the short-term and long-term vasopressin-mediated aquaporin-2 regulation by α-actinin 4, an F-actin crosslinking protein and a transcription co-activator of the glucocorticoid receptor. Vasopressin induced F-actin depolymerization and α-actinin 4 nuclear translocation in the mpkCCD collecting duct cell model. Co-immunoprecipitation followed by immunoblotting showed increased interaction between α-actinin 4 and glucocorticoid receptor in response to vasopressin. ChIP-PCR showed results consistent with α-actinin 4 and glucocorticoid receptor binding to the aquaporin-2 promoter. α-actinin 4 knockdown reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. α-actinin 4 knockdown did not affect vasopressin-induced glucocorticoid receptor nuclear translocation, suggesting independent mechanisms of vasopressin-induced nuclear translocation of α-actinin 4 and glucocorticoid receptor. Glucocorticoid receptor knockdown profoundly reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. In the absence of glucocorticoid analog dexamethasone, vasopressin-induced increases in glucocorticoid receptor nuclear translocation and aquaporin-2 mRNA were greatly reduced. α-actinin 4 knockdown further reduced vasopressin-induced increase in aquaporin-2 mRNA in the absence of dexamethasone. We conclude that glucocorticoid receptor plays a major role in vasopressin-induced aquaporin-2 gene expression that can be enhanced by α-actinin 4. In the absence of vasopressin, α-actinin 4 crosslinks F-actin underneath the apical plasma membrane, impeding aquaporin-2 membrane insertion. Vasopressin-induced F-actin depolymerization in one hand facilitates aquaporin-2 apical membrane insertion and in the other hand frees α-actinin 4 to enter the nucleus where it binds glucocorticoid receptor to enhance aquaporin-2 gene expression.