Fungal pathogens cause extensive plant diseases that damage crop production in the agricultural industry, resulting in annual crop loss, diminished food security, and historically significant epidemics. Though effective fungicides are available, their risks to the environment and animal health have increased the demand for more sustainable methods to control fungal pathogens. In plants, polygalacturonic-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromise plant cell walls and leave the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1, BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. We found that tPvPGIP2_5-8, which contains LRR5 to LRR8 and is of only one-third the size of the full-length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full length PvPGIP2 via Y2H. The inhibitory activities of tPvPGIP2_5-8 on the growth of A. niger were then examined and confirmed on pectin agar. Application of both full length PvPGIP2 and tPvPGIP2_5-8 clearly slows down the growth of A. niger and B. cinerea in the presence of pectin. The investigation on the sequence-function correlation of PvPGIP2 suggests that LRR5 could have the most essential structural feature for the inhibitory activities, and may be a possible target for the future engineering of PGIP with enhanced activity. This work highlights the potential of using plant-derived PGIPs as an exogenously applied fungal control agent both to plants and postharvest crops while minimally impacting the environment and human health.

In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1 and BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. We found that tPvPGIP2_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full length PvPGIP2 via Y2H. The inhibitory activities of tPvPGIP2_5-8 on the growth of A. niger and B. cinerea were then examined and confirmed on pectin agar. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 clearly slows down the growth of A. niger and B. cinerea. Investigation on the sequence-function relationships of PGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities, and may be a possible target for the future engineering of PGIP with enhanced activity. This study highlights the potential of plant-derived PGIPs as a candidate for future in planta evaluation as a pest control agent.

The COVID-19 crisis has taken a significant toll on human life and the global economy since its start in early 2020. Healthcare professionals have been particularly vulnerable because of the unprecedented shortage of Facepiece Respirators (FPRs), which act as fundamental tools to protect the medical staff treating the coronavirus patients. In addition, many FPRs are designed to be disposable single-use devices, creating an issue related to the generation of large quantities of non-biodegradable waste. In this contribution, we describe a plasma-based decontamination technique designed to circumvent the shortages of FPRs and alleviate the environmental problems posed by waste generation. The system utilizes a Dielectric Barrier Discharge (DBD) to generate ozone and feed it through the fibers of the FPRs. The flow-through configuration is different than canonical ozone-based sterilization methods, in which the equipment is placed in a sealed ozone-containing enclosure without any flow through the mask polymer fibers. We demonstrate the rapid decontamination of surgical masks using Escherichia coli (E. coli) and Vesicular Stomatitis Virus (VSV) as model pathogens, with the flow-through configuration providing a drastic reduction in sterilization time compared to the canonical approach. We also demonstrate that there is no deterioration in mask structure or filtration efficiency resulting from sterilization. Finally, we show that this decontamination approach can be implemented using readily available tools, such as a plastic box, a glass tube, few 3D printed components, and the high-voltage power supply from a plasma globe toy. The prototype assembled for this study is portable and affordable, with effectiveness comparable to that of larger and more expensive equipment.

During the beginning of 2020, the Covid-19 pandemic took the world by surprise, rapidly spreading undetected between and within many countries and wreaking havoc on the global economy both through death tolls and lockdowns. Healthcare professionals treating the coronavirus patients grapple with a massive and unprecedented shortage of Facepiece Respirators (FPRs) and other personal protective equipment (PPE), which act as fundamental tools to protect the health of the medical staff treating the patients affected by the coronavirus. While many FPRs are designed to be disposable single-use devices, the development of sterilization strategies is necessary to circumvent future shortages. Here, we describe the development of a plasma-based method to sterilize PPE such as FPRs with ozone. The novel design uses a flow-through configuration where ozone directly flows through the fibers of the PPE through the maintenance of a pressure gradient. Canonical ozone-based methods place the mask into a sealed ozone-containing enclosure but lack pressurization to permeate the mask fibers. In this device, ozone is created through an atmospheric pressure Dielectric Barrier Discharge (DBD) fed with compressed air. Due to limited supply and clinical need of FPRs, we demonstrated sterilization with surgical masks. We demonstrate rapid sterilization using E. coli as a model pathogen. A flow-through configuration enables a >400% improvement of the sterilization efficiency with respect to the canonical approach. This method has potential for a broad and cost-effective utilization. Using the power supply from a readily available plasma ball toy, a plastic box, a glass tube, steel mesh, and 3D printed components, we designed and tested an extremely affordable portable prototype system for rapid single mask sterilization which produced comparable results to its large high-cost equivalent.

Microbial chemical production is a rapidly growing industry, with much of the growth fueled by advances in synthetic biology. New approaches have enabled rapid strain engineering for the production of various compounds; however, translation to industry is often problematic because native phenotypes of model hosts prevent the design of new low-cost bioprocesses. Here, we argue for a new approach that leverages the native stress-tolerant phenotypes of non-conventional microbes that directly address design challenges from the outset. Growth at high temperature, high salt and solvent concentrations, and low pH can enable cost savings by reducing the energy required for product separation, bioreactor cooling, and maintaining sterile conditions. These phenotypes have the added benefit of allowing for the use of low-cost sugar and water resources. Non-conventional hosts are needed because these phenotypes are polygenic and thus far have proven difficult to recapitulate in the common hosts Escherichia coli and Saccharomyces cerevisiae .

The signal recognition particle (SRP) preferentially binds peptides destined for secretion before peptide-targeting signals are translated through recognition of elements in their mRNA, including non-coding sequences. As nascent proteins are generated by translating ribosomes, they are simultaneously targeted for translocation into the endoplasmic reticulum by a protein–RNA complex known as the signal recognition particle (SRP). Judith Frydman and co-workers were intrigued by how the SRP, which is in relatively low abundance, selects its substrates among a large number of nascent peptide chains emerging from ribosomes. Studying yeast cells, they find that the SRP preferentially binds substrates destined for secretion before they are fully translated — specifically, through the non-coding elements in their mRNA and before 'targeting signals' of these substrates are translated. Elsewhere in this issue of Nature, Günter Kramer and colleagues investigate the SRP interactome in the bacterium Escherichia coli and report that SRP almost exclusively targets hydrophobic transmembrane domains of inner membrane proteins, rejecting proteins of the outer membrane and the periplasmic space. Ribosome-associated factors must properly decode the limited information available in nascent polypeptides to direct them to their correct cellular fate1. It is unclear how the low complexity information exposed by the nascent chain suffices for accurate recognition by the many factors competing for the limited surface near the ribosomal exit site2,3. Questions remain even for the well-studied cotranslational targeting cycle to the endoplasmic reticulum, involving recognition of linear hydrophobic signal sequences or transmembrane domains by the signal recognition particle (SRP)4,5. Notably, the SRP has low abundance relative to the large number of ribosome–nascent-chain complexes (RNCs), yet it accurately selects those destined for the endoplasmic reticulum6. Despite their overlapping specificities, the SRP and the cotranslationally acting Hsp70 display precise mutually exclusive selectivity in vivo for their cognate RNCs7,8. To understand cotranslational nascent chain recognition in vivo, here we investigate the cotranslational membrane-targeting cycle using ribosome profiling9 in yeast cells coupled with biochemical fractionation of ribosome populations. We show that the SRP preferentially binds secretory RNCs before their targeting signals are translated. Non-coding mRNA elements can promote this signal-independent pre-recruitment of SRP. Our study defines the complex kinetic interaction between elongation in the cytosol and determinants in the polypeptide and mRNA that modulate SRP–substrate selection and membrane targeting.

Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C terminus, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 complex (Get4/5), which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3·Get4/5 complex. Here we report crystal structures of a Get3·Get4/5 complex from Saccharomyces cerevisiae at 2.8 and 6.0 Å that reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3·Get4/5 complex. Furthermore, we provide evidence that the Get3·Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limited electrostatically driven Get3·Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway.

Significance Quality control of proteins is critical to cellular homeostasis. The BCL2-associated athanogene cochaperone 6 (Bag6) complex, which contains Bag6, transmembrane domain recognition complex 35 (TRC35), and ubiquitin-like 4A (Ubl4A), plays an essential role in targeting transmembrane domains either to the endoplasmic reticulum or degradation. Bag6 is a central hub for numerous activities, functionally linked to an array of cellular pathways, from immunoregulation to apoptosis. Here we define the molecular architecture of this heterotrimer, revealing distinct binding sites on Bag6 for TRC35 and Ubl4A. The truncated Bag6 complex defined in this study is sufficient to facilitate substrate transfer from small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) to TCR40. In addition, structural and biochemical characterization of the BAG domain of Bag6 demonstrate that it is not a canonical BAG domain.

Correct localization of membrane proteins is essential to all cells. Chaperone cascades coordinate the capture and handover of substrate proteins from the ribosomes to the target membranes, yet the mechanistic and structural details of these processes remain unclear. Here we investigate the conserved GET pathway, in which the Get4-Get5 complex mediates the handover of tail-anchor (TA) substrates from the cochaperone Sgt2 to the Get3 ATPase, the central targeting factor. We present a crystal structure of a yeast Get3-Get4-Get5 complex in an ATP-bound state and show how Get4 primes Get3 by promoting the optimal configuration for substrate capture. Structure-guided biochemical analyses demonstrate that Get4-mediated regulation of ATP hydrolysis by Get3 is essential to efficient TA-protein targeting. Analogous regulation of other chaperones or targeting factors could provide a general mechanism for ensuring effective substrate capture during protein biogenesis.

Parkinson's disease (PD) is a common neurodegenerative disorder. Functional interactions between some PD genes, like PINK1 and parkin, have been identified, but whether other ones interact remains elusive. Here we report an unexpected genetic interaction between two PD genes, VPS35 and EIF4G1. We provide evidence that EIF4G1 upregulation causes defects associated with protein misfolding. Expression of a sortilin protein rescues these defects, downstream of VPS35, suggesting a potential role for sortilins in PD. We also show interactions between VPS35, EIF4G1, and α-synuclein, a protein with a key role in PD. We extend our findings from yeast to an animal model and show that these interactions are conserved in neurons and in transgenic mice. Our studies reveal unexpected genetic and functional interactions between two seemingly unrelated PD genes and functionally connect them to α-synuclein pathobiology in yeast, worms, and mouse. Finally, we provide a resource of candidate PD genes for future interrogation. VIDEO ABSTRACT:

The genetic code allows most amino acids a choice of optimal and nonoptimal codons. We report that synonymous codon choice is tuned to promote interaction of nascent polypeptides with the signal recognition particle (SRP), which assists in protein translocation across membranes. Cotranslational recognition by the SRP in vivo is enhanced when mRNAs contain nonoptimal codon clusters 35-40 codons downstream of the SRP-binding site, the distance that spans the ribosomal polypeptide exit tunnel. A local translation slowdown upon ribosomal exit of SRP-binding elements in mRNAs containing these nonoptimal codon clusters is supported experimentally by ribosome profiling analyses in yeast. Modulation of local elongation rates through codon choice appears to kinetically enhance recognition by ribosome-associated factors. We propose that cotranslational regulation of nascent-chain fate may be a general constraint shaping codon usage in the genome.

Tail‐anchored (TA) membrane proteins are targeted to the endoplasmic reticulum via the post‐translational GET pathway. A critical step in this essential pathway is the transfer of TA substrates from the co‐chaperone Sgt2 to the Get3 ATPase, mediated by the hetero‐tetrameric complex Get4/Get5 (Get4/5). However, a major outstanding question is how Get4 binds to Get3 and regulates its activity. Here we report a 5.4 Å crystal structure of an ATP‐bound Get3‐Get4/5 complex from Saccharomyces cerevisiae (Sc), the largest pathway components solved to date. Our structure reveals the role of nucleotide in complex formation and the presence of two functionally distinct binding interfaces. Mutational analysis confirms that one interface is required for the specificity of binding, while the other interface is involved in the regulation of Get3 ATPase activity. Additional functional assays demonstrate that Get4/5‐mediated regulation of ATP hydrolysis by Get3 is essential for efficient TA protein targeting. Our work also supports a model for Get3/TA complex formation in which two opposing Get3 dimers are bridged by a single Get4/5 hetero‐tetramer. Finally, we present a 2.8 Å crystal structure of an apo ScGet3‐Get4/5 complex that provides evidence for a rapid initial binding state mediated by electrostatics. This work illustrates how Get4/5 regulates Get3, priming it for TA loading, a critical step in this important pathway.Grant Funding Source: NIH

Tail-anchored trans-membrane proteins are targeted to membranes post-translationally. The proteins Get4 and Get5 form an obligate complex that catalyzes the transfer of tail-anchored proteins destined to the endoplasmic reticulum from Sgt2 to the cytosolic targeting factor Get3. Get5 forms a homodimer mediated by its carboxyl domain. We show here that a conserved motif exists within the carboxyl domain. A high resolution crystal structure and solution NMR structures of this motif reveal a novel and stable helical dimerization domain. We additionally determined a solution NMR structure of a divergent fungal homolog, and comparison of these structures allows annotation of specific stabilizing interactions. Using solution x-ray scattering and the structures of all folded domains, we present a model of the full-length Get4/Get5 complex.

Biosynthesis of membrane proteins requires that hydrophobic transmembrane (TM) regions be shielded from the cytoplasm while being directed to the correct membrane. Tail-anchored (TA) membrane proteins, characterized by a single C-terminal TM, pose an additional level of complexity because they must be post-translationally targeted. In eukaryotes, the GET pathway shuttles TA-proteins to the endoplasmic reticulum. The key proteins required in yeast (Sgt2 and Get1-5) have been under extensive structural and biochemical investigation during recent years. The central protein Get3 utilizes nucleotide linked conformational changes to facilitate substrate loading and targeting. Here we analyze this complex process from a structural perspective, as understood in yeast, and further postulate on similar pathways in other domains of life.

Tail-anchored (TA) proteins are ubiquitous in eukaryotes and fulfill various important functions in the cell, such as the mediation of cell-cell contact and apoptosis signaling. Because they are anchored to the membrane by a short transmembrane domain (TMD) at the C-terminus, they cannot utilize the cotranslational signal recognition particle (SRP) pathway. Recent biochemical and structural studies have helped us identify and elucidate the details of the TA protein targeting pathway in yeast, also known as the Guided Entry of TA protein (GET) pathway. A homologous pathway, the TMD Recognition Complex (TRC) pathway, has been identified but details of this pathway remain elusive. In this study, we have structurally and biochemically characterized Bag6 and Ubl4Apreviously shown to be crucial in the recognition and delivery TA proteins from the ribosome to TRC40.

Tail‐anchored (TA) proteins are classified by a single transmembrane helix at the extreme carboxyl terminus. In yeast, the Guided Entry of Tail‐anchored proteins (GET) pathway facilitates the post‐translational targeting of TA proteins to the endoplasmic reticulum (ER). A complex consisting of Get4, Get5, Sgt2 and chaperones delivers ER destined substrates to Get3, which then targets the substrate to the ER membrane. We have structurally characterized the Get4/Get5/Sgt2 complex. Get4 is an alpha‐helical repeat protein that directly binds Get3. Get4 and Get5 form an obligate dimer mediated by the amino terminal domain of Get5, and this heterodimer forms a higher order dimer mediated by the carboxyl terminal domain of Get5. We have determined structures of the Get5 homodimerization domain by crystallography and solution NMR and found it is a novel, high‐affinity oligomerization motif. Sgt2 contains a tetratricopeptide repeat (TPR) domain that binds to a wide variety of heat shock cognate (HSC) protein families. A crystal structure of the TPR domain with an HSC‐analog provides an explanation for this low specificity. Sgt2 also contains an amino terminal homodimerization domain that additionally binds a ubiquitin‐like domain within Get5. We utilize bioSAXS to combine high‐resolution structural data, generating a model of the Get4/Get5/Sgt2 complex in solution.