Synthesis Of Membrane Proteins Research Articles

Novel protein trafficking and signaling pathways in kidney physiology and pathophysiology

The nephron's fluid and electrolyte transport properties are determined largely by the inventories of ion transport proteins occupying the apical and basolateral plasma membrane domains of renal tubule epithelial cells. The asymmetric apportioning of ion channels, transporters and pumps among these domains determines the nature of the fluxes maintained by renal epithelia. Generation and maintenance of differentiated plasmalemmal domains requires a cell to possess machinery capable of discriminating among newly synthesized membrane proteins. Information embedded in these proteins' structures serves as sorting signals that specify their appropriate destinations.To follow the fates of newly synthesized membrane proteins in polarized epithelial cells, we have adapted a technique that permits direct observation of temporally defined cohorts of proteins via the combination of fluorescence microscopy with pulse‐chase labeling protocols. Using this technique, we can follow cohorts of protein as they are synthesized and trafficked. We have found that different proteins that are destined for the same destination appear to travel to the cell surface in separate vesicular carriers. We have also found that the base of the primary cilium appears to be a “hot spot” for the delivery of newly synthesized proteins.Autosomal Dominant Polycystic Kidney Disease (ADPKD) affects ~1 in 1,000 people and is the most common potentially lethal genetic condition, leading to end stage renal disease in ~50% of patients. ADPKD is caused by mutations in the genes encoding polycystin‐1 (PC1) and polycystin‐2 (PC2). PC1 is an enormous membrane protein that spans the bilayer 11 times, with a large extracellular N terminal domain and a short C terminal domain that faces the cytoplasm. PC2 is a member of the TRP family of non‐selective calcium‐permeable cation channels. The PC1 and PC2 proteins participate in a number of signaling pathways and exhibit complex patterns of subcellular localization. PC1 and PC2 interact with each other and with numerous other proteins that may modulate their trafficking properties or their involvement in signal transduction. Both PC1 and PC2 localize to the primary cilium where they appear to participate in mechanosensory or chemosensory processes. We have found that polycystin trafficking and function are regulated by oxygen‐sensing pathways. We have also found that the C‐terminal tail of Polycystin‐1 is released by a proteolytic cleavage, allowing it to travel to the nucleus, where it is involved in the regulation of several transcriptional pathways that modulate proliferation, apoptosis and cell fate determination. Finally, we find that PC1 functions as a receptor in a novel signal transduction pathway that regulates cell growth.Support or Funding InformationNIDDK and DoDThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The intramembrane protease SPP impacts morphology of the endoplasmic reticulum by triggering degradation of morphogenic proteins

The endoplasmic reticulum (ER), as a multifunctional organelle, plays crucial roles in lipid biosynthesis and calcium homeostasis as well as the synthesis and folding of secretory and membrane proteins. Therefore, it is of high importance to maintain ER homeostasis and to adapt ER function and morphology to cellular needs. Here, we show that signal peptide peptidase (SPP) modulates the ER shape through degradation of morphogenic proteins. Elevating SPP activity induces rapid rearrangement of the ER and formation of dynamic ER clusters. Inhibition of SPP activity rescues the phenotype without the need for new protein synthesis, and this rescue depends on a pre-existing pool of proteins in the Golgi. With the help of organelle proteomics, we identified certain membrane proteins to be diminished upon SPP expression and further show that the observed morphology changes depend on SPP-mediated cleavage of ER morphogenic proteins, including the SNARE protein syntaxin-18. Thus, we suggest that SPP-mediated protein abundance control by a regulatory branch of ER-associated degradation (ERAD-R) has a role in shaping the early secretory pathway.

Functional and Structural Analysis of Cell-Free Synthesized Membrane Proteins

Functional and Structural Analysis of Cell-Free Synthesized Membrane Proteins

MGSE Regulates Crosstalk from the Mucin Pathway to the TFE3 Pathway of the Golgi Stress Response.

The Golgi apparatus is an organelle where membrane or secretory proteins receive post-translational modifications such as glycosylation and sulfation, after which the proteins are selectively transported to their final destinations through vesicular transport. When the synthesis of secretory or membrane proteins is increased and overwhelms the capacity of the Golgi (Golgi stress), eukaryotic cells activate a homeostatic mechanism called the Golgi stress response to augment the capacity of the Golgi. Four response pathways of the Golgi stress response have been identified, namely the TFE3, CREB3, HSP47, and proteoglycan pathways, which regulate the general function of the Golgi, apoptosis, cell survival, and proteoglycan glycosylation, respectively. Here, we identified a novel response pathway that augments the expression of glycosylation enzymes for mucins in response to insufficiency in mucin-type glycosylation in the Golgi (mucin-type Golgi stress), and we found that expression of glycosylation enzymes for mucins such as GALNT5, GALNT8, and GALNT18 was increased upon mucin-type-Golgi stress. We named this pathway the mucin pathway. Unexpectedly, mucin-type Golgi stress induced the expression and activation of TFE3, a key transcription factor regulating the TFE3 pathway, suggesting that the activated mucin pathway sends a crosstalk signal to the TFE3 pathway. We identified an enhancer element regulating transcriptional induction of TFE3 upon mucin-type Golgi stress, and named it the mucin-type Golgi stress response element, of which consensus was ACTTCC(N9)TCCCCA. These results suggested that crosstalk from the mucin pathway to the TFE3 pathway has an important role in the regulation of the mammalian Golgi stress response.Key words: Golgi stress, mucin, TFE3, organelle autoregulation, organelle zone.

Membrane Protein Sorting: Proteoliposome Engineering with Cell‐Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems (Adv. Sci. 10/2018)

Membrane Protein Sorting: Proteoliposome Engineering with Cell‐Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems (Adv. Sci. 10/2018)

Investigation of the Molecular Mechanisms Leading to Protein Translation Inhibition in Response to Endoplasmic Reticulum Stress

Protein folding is an important process for cellular function. ER is responsible for the synthesis, folding, modification, and quality control of numerous secretory and membrane proteins. The intracellular disturbance caused by different stressors leading to the accumulation of unfolded/ misfolded proteins can all lead to an alteration in ER homeostasis. If the unfolded/misfolded proteins continue to accumulate inside the ER, the unfolded protein response (UPR) is induced to overcome this situation. UPR acts by three different mechanisms:(1) increase the ER protein-folding capacity (Robertsonand Branch, 1987),(2) reduce global protein synthesis, and (3) enhance ER-associated degradation process. The UPR is mediated by three ER transmembrane protein sensors: (1) activating transcription factor 6 (ATF6), (2) inositol requiring kinase 1(IRE1), and (3) double-stranded RNA-activated protein kinase (PKR) like endoplasmic reticulum kinase (PERK). Each sensor of the UPR protein responds to the certain level of unfolded/misfolded protein in the ER. If the cell fails to restore or overcome the protein-folding defect, cell-death signaling pathways are activated.

Proteoliposome Engineering with Cell-Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems.

Integral membrane proteins (IMPs) modulate key cellular processes; their dysfunctions are closely related to disease. However, production of IMPs in active conformations for further study is hindered by aggregation and toxicity in living expression systems. IMPs are therefore produced in cell‐free systems employing liposome chaperoning, but membrane integration of the nascent IMPs is suboptimal and orientation of the integrated proteins remains uncontrollable. Thus, an artificial membrane protein sorting system is developed, based on polyhistidine/nickel‐chelate affinity, combined with cell‐free membrane protein synthesis. Its proof of concept is demonstrated with a N‐terminal hexahistadine‐fused conexin‐43 (NHis–Cx43) model IMP. Nickel‐chelating liposomes efficiently incorporate twofold newly synthesized NHis–Cx43 compared with Cx43. NHis–Cx43, when synthesized in this system, forms dye‐permeable hemichannels, similar to plasma membrane pores formed by Cx43 in cells. The topology of incorporated NHis–Cx43 indicates two orientations in the liposomal membranes. However, NHis–Cx43 orientation is controlled, resulting in single topology, by combination of the natural molecular chaperone DnaKJE. Successful synthesis and at least 4.5‐fold increase lipid incorporation are also achieved with three other NHis‐fused IMPs, including α‐helix and β‐barrel IMPs. Overall, this simple membrane protein sorting system is usable combined with molecular chaperones to prepare proteoliposomes for many applications.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane

The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E.coli surface, with a nearest neighbor distance of ∼200nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.

A Transcriptomic Analysis of Saccharomyces cerevisiae Under the Stress of 2-Phenylethanol

2-Phenylethanol (2-PE) is a kind of advanced aromatic alcohol with rose fragrance, which is wildly used for the deployment of flavors and fragrances. Microbial transformation is the most feasible method for the production of natural 2-PE. But a bottleneck problem is the toxicity of 2-PE on the cells. The molecular mechanisms of the toxic effect of 2-PE to Saccharomyces cerevisiae are not well studied. In this study, we analyzed the transcriptomes of S. cerevisiae in the media with and without 2-PE, respectively, using Illumina RNA-Seq technology. We identified 580 differentially expressed genes between S. cerevisiae in two different treatments. GO and KEGG enrichment analyses of these genes suggested that most genes encoding mitochondrial proteins, cytoplasmic, and plasma membrane proteins were significantly up-regulated, whereas the enzymes related to amino acid metabolism were down-regulated. These results indicated that 2-PE suppressed the synthesis of plasma membrane proteins, which suppressed the transport of nutrients required for growth. The findings in this study will provide insight into the inhibitory mechanism of 2-PE to yeast and other microbes.

Unbiased Proteomic Study of the Axons of Cultured Rat Cortical Neurons.

The axon is a long projection connecting a neuron to its targets. Here, the axons of cultured rat cortical neurons were isolated with micropatterned chips that enable the separation of axons from their cell bodies. Proteins extracted from isolated axons and whole neurons were subjected to analyses using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) analyses without and with stable isotope dimethyl labeling, resulting in the identification of >2500 axonal proteins and 103 axon-enriched proteins. A strong correlation exists between the abundances of axonal proteins and their counterparts in whole neurons. The proteomic results confirm the axonal protein constituents of the subcellular structures documented in earlier electron microscopic studies. Cortical axons have proteins that are components of machineries for protein degradation and the synthesis of soluble, membrane, and secretory proteins, although axons lack conventional Golgi apparatus. Despite the fact that axons lack nucleus, nuclear proteins were identified, and 67 of them were found enriched in axons. Some of the results obtained by the MS-based studies were validated by quantitative Western blotting and immunofluorescence staining analyses. The results represent the first comprehensive description of the axonal protein landscape. The MS proteomics data are available via ProteomeXchange with identifier PXD005527.

Exogenous spermidine maintains the chloroplast structure of cucumber seedlings and inhibits the degradation of photosynthetic protein complexes under high-temperature stress

Cucumbers (Cucumis sativus L.) are thermophilic horticulture crop but do not tolerate high temperature. The photosynthesis of cucumber under high temperature is highly susceptible to damage. To study the physiological mechanism of exogenous spermidine (Spd) on cucumber photosynthesis under heat stress, the sensitive cucumber cultivar ‘Jinchun no. 2’ was grown in substrate culture at a high temperature in an artificial climate box and treated with 1.0 mmol L−1 Spd under high-temperature stress (42/32 °C). The results showed that exogenous Spd alleviated the photosynthetic damage caused by heat shock, added the chlorophyll content and maintained the chloroplast structures relatively intact. Western blotting analysis showed that exogenous Spd inhibited the degradation of photosynthetic proteins, slowed the dissociation of the protein complexes, and maintained the stability of light-harvesting complexes (LHCs) in cucumber leaves under high-temperature stress. Twenty-two differentially expressed thylakoid membrane proteins involved in different photosynthetic processes were successfully authenticated by sodium dodecyl sulphate (SDS)-urea-PAGE and Blue native-polyacrylamide gel electrophoresis (BN-PAGE). The results showed that exogenous Spd regulated the expression of photosynthetic and bursal somatic membrane proteins, which resulted in adaptive changes under high-temperature stress at the transcriptional and translational levels, inhibited the degradation of thylakoid membrane proteins in cucumber leaves, and maintained the stable structure of the thylakoid membrane. Collectively, these results suggested that exogenous Spd alleviated high temperature-induced photosynthesis damage by improving the expression and synthesis of thylakoid membrane proteins, mitigating the dissociation of LHCII–Chl and thylakoid membrane protein complexes and maintaining the integrity and functional stability of the photosynthetic organ structure.

Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

Diel regulation of photosynthetic activity in the oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501.

The oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501 exhibits large diel changes in abundance of both Photosystem II (PSII) and Photosystem I (PSI). To understand the mechanisms underlying these dynamics, we assessed photosynthetic parameters, photosystem abundance and composition, and chlorophyll-protein biosynthesis over a diel cycle. Our data show that the decline in PSII activity and abundance observed during the dark period was related to a light-induced modification of PSII, which, in combination with the suppressed synthesis of membrane proteins, resulted in monomerization and gradual disassembly of a large portion of PSII core complexes. In the remaining population of assembled PSII monomeric complexes, we detected the non-functional version of the D1 protein, rD1, which was absent in PSII during the light phase. During the dark period, we also observed a significant decoupling of phycobilisomes from PSII and a decline in the chlorophyll a quota, which matched the complete loss of functional PSIIs and a substantial decrease in PSI abundance. However, the remaining PSI complexes maintained their photochemical activity. Thus, during the nocturnal period of nitrogen fixation C. watsonii operates a suite of regulatory mechanisms for efficient utilization/recycling of cellular resources and protection of the nitrogenase enzyme.

Chemical synthesis of membrane proteins: a model study on the influenza virus B proton channel.

In the present study we have developed and optimized a robust strategy for the synthesis of highly hydrophobic peptides, especially membrane proteins, exemplarily using the influenza B M2 proton channel (BM2(1-51)). This strategy is based on the native chemical ligation of two fragments, where the thioester fragment is formed from an oxo-ester peptide, which is synthesized using Fmoc-SPPS, and features an in situ cleavable solubilizing tag (ADO, ADO2 or ADO-Lys5). The nearly quantitative production of the ligation product was followed by an optimized work up protocol, resulting in almost quantitative desulfurization and Acm-group cleavage. Circular dichroism analysis in a POPC lipid membrane revealed that the synthetic BM2(1-51) construct adopts a helical structure similar to that of the previously characterized BM2(1-33).

Quantitative analysis of cell-free synthesized membrane proteins at the stabilized droplet interface bilayer

We report the functional synthesis and quantification of membrane proteins-α-hemolysin from Staphylococcus aureus and the multidrug transporter EmrE from Escherichia coli-at the stabilized droplet interface bilayer using an in vitro transcription-translation system. The system developed here can expand the list of integral membrane proteins applicable for quantitative functional analysis.

Comparative Proteomics and Functional Analysis of Red Blood Cell Membrane Proteins in the Rare in(Lu) Blood Type

KLF1 is an essential erythroid transcription factor (EKLF) involved both in the ß-globin switch from fetal to adult and in definitive erythropoiesis. KLF1 also activates genes encoding heme biosynthetic enzymes, as well as genes involved in the cell cycle and the synthesis of membrane proteins. Heterozygosity for one or several nucleotide changes in KLF1 may be responsible for the so-called dominant Lu(a-b-) phenotype, also known as In(Lu) for “Inhibitor Lutheran” and characterized by a dramatic reduction in the expression levels of the Lutheran blood group antigens and a slight elevation in hemoglobin F (HbF). Several other blood group antigens are also under the direct control of KLF1, but their number and expression level in In(Lu) red blood cells (RBCs) remain a matter of debate. In addition, mutations in KLF1 in humans lead to anemia, acantocytosis and some of these variants, such as CDA type IV, are characterized by ineffective terminal differentiation and defects in enucleation. Given the major role of KLF1 in erythropoiesis, it would not be surprising that In(Lu) RBCs show abnormal properties. In this work, we performed proteomic studies to quantify membrane proteins in In(Lu) vs control RBCs.Results: using label-free mass spectrometry, we analyzed the expression levels of membrane proteins in 5 In(Lu) and 4 control RBCs. Hierarchical clustering allowed to identify two modulation profiles of protein expression (Figure 1). The first profile (red color) is composed of 49 proteins over-expressed in In(Lu) RBCs, with the majority of them corresponding to the “26S proteasome complex” (PSMA, PSMB, PSMD, and PSME). In addition, we confirmed the overexpression of the 26S proteasome complex in In(Lu) RBCs by western blot analyses.The second profile (green color) includes 23 membrane proteins with a lower expression level in In(Lu) RBCs; these proteins correspond to blood group antigens (e.g. Lu/BCAM, CD44), and to cytoskeletal proteins (e.g. dematin, flotillin). Five proteins carrying blood group systems show a decrease of expression in In(Lu) RBCs over 40%: Chido/Rodgers, Xg, Indian, Duffy and Lutheran: antigens. While our results indicating of decreased expression of Indian and Chido/Rodgers systems are consistent with previous reports, the Duffy expression was unexpectedly found to be decreased by 86%.Since we evidenced an important increase in the proteasome complex, and taking into account that KLF1 is involved in the regulation or erythropoiesis, we, investigated the expression profile of the 26 S proteasome expression during erythropoiesis. We observed an important decrease of all proteins of the proteasome complex during erythropoiesis.Conclusion: from our preliminary results reported in this study, we hypothesized that the increase of the 26S proteasome in the In(Lu) individuals may result from defects in organelle loss and could explain the profound dyserytropoiesis observed in the “nan” mice null for KLF1.Figure 1: Comparative proteomics of red blood cell membrane proteins in In(Lu) blood group type. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Protein Targeting: ER Leads the Way to the Inner Nuclear Envelope

Efficient targeting of newly synthesized membrane proteins from the endoplasmic reticulum to the inner nuclear membrane depends on nucleotide hydrolysis. A new study shows that this dependence reflects critical actions of the atlastin family of GTPases in maintaining the morphology of the endoplasmic reticulum network.

Chemical synthesis of membrane proteins by the removable backbone modification method

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation.

Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.

Signal recognition particle binds to translating ribosomes before emergence of a signal anchor sequence.

The bacterial signal recognition particle (SRP) is part of the machinery that targets ribosomes synthesizing membrane proteins to membrane-embedded translocons co-translationally. Recognition of nascent membrane proteins occurs by virtue of a hydrophobic signal-anchor sequence (SAS) contained in the nascent chain, usually at the N terminus. Here we use fluorescence-based stopped-flow to monitor SRP-ribosome interactions with actively translating ribosomes while an SRP substrate is synthesized and emerges from the peptide exit tunnel. The kinetic analysis reveals that, at cellular concentrations of ribosomes and SRP, SRP rapidly binds to translating ribosomes prior to the emergence of an SAS and forms an initial complex that rapidly rearranges to a more stable engaged complex. When the growing peptide reaches a length of ∼50 amino acids and the SAS is partially exposed, SRP undergoes another conformational change which further stabilizes the complex and initiates targeting of the translating ribosome to the translocon. These results provide a reconciled view on the timing of high-affinity targeting complex formation, while emphasizing the existence of preceding SRP recruitment steps under conditions of ongoing translation.