Vacuolar Protease Research Articles

Marker-free genomic editing in Saccharomyces cerevisiae using universal donor templates and multiplexing CRISPR-CAS9.

The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying a variety of critical cellular processes. Traditional methods to knock in or -out at specific yeast loci utilize polymerase chain reaction-based techniques, in which marker cassettes with gene-specific homologies are integrated into the genome via homologous recombination. While simple and cost-effective, these methods are limited by marker availability when multiple edits are desired. More recently, CRISPR-Cas9 technology has introduced methods to edit the yeast genome without the need for selectable markers. Although efficient, this method is hindered by additional reagents and lengthy protocols to design and test unique guide RNAs and donor templates for each desired edit. In this study, we have combined these two approaches and have developed a highly efficient economical method to edit the yeast genome marker-free. We have designed two universal donor templates that efficiently repair commonly used selectable markers when targeted by a novel guideRNA-Cas9 designed to promoter regions in Ashbya gossypii found in most integration modules. Furthermore, we find our newly designed guideRNA-Cas9 successfully multiplexes when multiple markers are present. Using these new tools, we have significantly improved the cost and efficiency to generate single or multiple marker-free genetic modifications. In this study, we demonstrate the effectiveness of these new tools by marker-free ablating PRC1, PEP4, and PRB1 vacuolar proteases typically inactivated before many biochemical and membrane-trafficking studies using budding yeast.

The mechanism of Atg15-mediated membrane disruption in autophagy.

Autophagy is a lysosomal/vacuolar delivery system that degrades cytoplasmic material. During autophagy, autophagosomes deliver cellular components to the vacuole, resulting in the release of a cargo-containing autophagic body (AB) into the vacuole. AB membranes must be disrupted for degradation of cargo to occur. The lipase Atg15 and vacuolar proteases Pep4 and Prb1 are known to be necessary for this disruption and cargo degradation, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and show that Atg15 is the sole vacuolar phospholipase. Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments reveal that Atg15 is a phospholipase B of broad substrate specificity that is likely implicated in the disruption of a range of membranes. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.

Topology and Function of the S. cerevisiae Autophagy Protein Atg15.

The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into the MVB pathway. By replacing this domain, we generated chimeras located in the cytosol, the vacuole membrane, and the lumen. The variants at the vacuole membrane and in the lumen were highly active. Together with the absence of Atg15 from the phagophore and autophagic bodies, this suggests that, within the vacuole, Atg15 can lyse vesicles where it is not embedded. In-depth topological analyses showed that Atg15 is a single membrane-spanning protein with the amino-terminus in the cytosol and the rest, including the active site motif, in the ER lumen. Remarkably, only membrane-embedded Atg15 variants affected growth when overexpressed. The growth defects depended on its active site serine 332, showing that it was linked to the enzymatic activity of Atg15. Interestingly, the growth defects were independent of vacuolar proteinase A and vacuolar acidification.

Assembly of selenium nanoparticles by protein coronas composed of yeast protease A

The biosafety profile of protein corona-encapsulated selenium nanoparticles (SeNPs) has stimulated great interest among biomedical and therapeutic researchers. However, elucidating the potential mechanisms underlying the assembly process remains a significant challenge. Herein, we synthesized protein corona-encapsulated SeNPs using Saccharomyces boulardii, and then systematically investigated the complex constituents. The corona-forming protein constituents were analyzed after separation and purification. Protein sequencing results confirmed that PEP4p vacuolar aspartyl protease (protease A, PrA) participates in the SeNP-encapsulating protein corona assembly process. Additionally, protein- encapsulated SeNPs were produced using wild-type and mutant strains to determine the potential mechanism of corona formation. Phylogenetic analysis showed that the mutant strain promoted the accumulation of SeNPs and facilitated extensive production of protein corona-SeNP complexes, which is a universal cellular stress response among yeasts. This study provides an important theoretical and practical basis for the synthesis of various protein corona-coated NP complexes. The protein corona-encapsulated SeNPs fell within an acceptable size range and were effectively protein coated, indicating their potential for nutritional and pharmaceutical applications for humans and animals.

Overexpression of a Novel Vacuolar Serine Protease-Encoding Gene (spt1) to Enhance Cellulase Production in Trichoderma Reesei

Trichoderma reesei is widely applied as the major industrial fungus for the production of cellulases used for the conversion of lignocellulosic biomass to biofuels and other biobased products. The protein secretion pathway is vital for cellulase secretion, but few reports are related to the role of the vacuole in cellulase production. Here, we identified a novel vacuolar serine protease gene spt1 and investigated the ability of T. reesei to secrete cellulases by disrupting, complementing and overexpressing the spt1 gene. Amino acid sequence analysis of the Spt1 protein showed that it belongs to the subtilisin S8 family and has the conserved catalytic triples (Asp, His, Ser) of the serine protease. The deletion of spt1 did not lead to a decrease in extracellular protease activity, and the observation of mycelia with the Spt1–eGFP fusion expression and the vacuolar membrane dye FM4-64 staining confirmed that Spt1 was an intracellular protease located in the vacuoles of T. reesei. However, the spt1 gene deletion significantly reduced spore production and cellulase secretion, while the spt1 complementation recovered these traits to those of the parental strain. When spt1 was overexpressed by using its native promoter and introducing multiple copies, the cellulase secretion was improved. Furthermore, a strong promoter, Pcdna1, was used to drive the spt1 overexpression, and it was found that the cellulase production was significantly enhanced. Specifically, the filter paper activity of the spt1 overexpression strain SOD-2 reached 1.36 U/mL, which was 1.72 times higher than that of the parental strain. These findings demonstrated that the spt1 gene can be a powerful target for increasing cellulase production in T. reesei, which suggests a possible important role of the vacuole in the cellulase secretion pathway and provides new clues for improving strains for efficient cellulase production.

Vacuolar Processing Enzymes in Plant Programmed Cell Death and Autophagy.

Vacuolar processing enzymes (VPEs) are plant cysteine proteases that are subjected to autoactivation in an acidic pH. It is presumed that VPEs, by activating other vacuolar hydrolases, are in control of tonoplast rupture during programmed cell death (PCD). Involvement of VPEs has been indicated in various types of plant PCD related to development, senescence, and environmental stress responses. Another pathway induced during such processes is autophagy, which leads to the degradation of cellular components and metabolite salvage, and it is presumed that VPEs may be involved in the degradation of autophagic bodies during plant autophagy. As both PCD and autophagy occur under similar conditions, research on the relationship between them is needed, and VPEs, as key vacuolar proteases, seem to be an important factor to consider. They may even constitute a potential point of crosstalk between cell death and autophagy in plant cells. This review describes new insights into the role of VPEs in plant PCD, with an emphasis on evidence and hypotheses on the interconnections between autophagy and cell death, and indicates several new research opportunities.

Processing of Fluorescent Proteins May Prevent Detection of Prion Particles in [PSI+] Cells.

Yeast is a convenient model for studying protein aggregation as it is known to propagate amyloid prions. [PSI+] is the prion form of the release factor eRF3 (Sup35). Aggregated Sup35 causes defects in termination of translation, which results in nonsense suppression in strains carrying premature stop codons. N-terminal and middle (M) domains of Sup35 are necessary and sufficient for maintaining [PSI+] in cells while preserving the prion strain's properties. For this reason, Sup35NM fused to fluorescent proteins is often used for [PSI+] detection and investigation. However, we found that in such chimeric constructs, not all fluorescent proteins allow the reliable detection of Sup35 aggregates. Particularly, transient overproduction of Sup35NM-mCherry resulted in a diffuse fluorescent pattern in the [PSI+] cells, while no loss of prions and no effect on the Sup35NM prion properties could be observed. This effect was reproduced in various unrelated strain backgrounds and prion variants. In contrast, Sup35NM fused to another red fluorescent protein, TagRFP-T, allowed the detection of [PSI+] aggregates. Analysis of protein lysates showed that Sup35NM-mCherry is actively degraded in the cell. This degradation was not caused by vacuolar proteases and the ubiquitin-proteasomal system implicated in the Sup35 processing. Even though the intensity of this proteolysis was higher than that of Sup35NM-GFP, it was roughly the same as in the case of Sup35NM-TagRFP-T. Thus, it is possible that, in contrast to TagRFP-T, degradation products of Sup35NM-mCherry still preserve their fluorescent properties while losing the ability to decorate pre-existing Sup35 aggregates. This results in diffuse fluorescence despite the presence of the prion aggregates in the cell. Thus, tagging with fluorescent proteins should be used with caution, as such proteolysis may increase the rate of false-negative results when detecting prion-bearing cells.

Vacuolar proteases and autophagy in phytopathogenic fungi: A review.

Autophagy (macroautophagy) is a survival and virulence mechanism of different eukaryotic pathogens. Autophagosomes sequester cytosolic material and organelles, then fuse with or enter into the vacuole or lysosome (the lytic compartment of most fungal/plant cells and many animal cells, respectively). Subsequent degradation of cargoes delivered to the vacuole via autophagy and endocytosis maintains cellular homeostasis and survival in conditions of stress, cellular differentiation, and development. PrA and PrB are vacuolar aspartyl and serine endoproteases, respectively, that participate in the autophagy of fungi and contribute to the pathogenicity of phytopathogens. Whereas the levels of vacuolar proteases are regulated by the expression of the genes encoding them (e.g., PEP4 for PrA and PRB1 for PrB), their activity is governed by endogenous inhibitors. The aim of the current contribution is to review the main characteristics, regulation, and role of vacuolar soluble endoproteases and Atg proteins in the process of autophagy and the pathogenesis of three fungal phytopathogens: Ustilago maydis, Magnaporthe oryzae, and Alternaria alternata. Aspartyl and serine proteases are known to participate in autophagy in these fungi by degrading autophagic bodies. However, the gene responsible for encoding the vacuolar serine protease of U. maydis has yet to be identified. Based on in silico analysis, this U. maydis gene is proposed to be orthologous to the Saccharomyces cerevisiae genes PRB1 and PBI2, known to encode the principal protease involved in the degradation of autophagic bodies and its inhibitor, respectively. In fungi that interact with plants, whether phytopathogenic or mycorrhizal, autophagy is a conserved cellular degradation process regulated through the TOR, PKA, and SNF1 pathways by ATG proteins and vacuolar proteases. Autophagy plays a preponderant role in the recycling of cell components as well as in the fungus-plant interaction.

Homology Modeling and Analysis of Vacuolar Aspartyl Protease from a Novel Yeast Expression Host Meyerozyma guilliermondii Strain SO

Homology Modeling and Analysis of Vacuolar Aspartyl Protease from a Novel Yeast Expression Host Meyerozyma guilliermondii Strain SO

Monoubiquitinated MxIRT1 acts as an iron receptor to determine MxIRT1 vacuole degradation or plasma membrane recycling via endocytosis

ABSTRACT IRON-REGULATED TRANSPORTER 1 (IRT1) is critical for iron uptake in roots, and its exocytosis to the plasma membrane (PM) is regulated by the iron status sensed by the histidine-rich domain (HRM). However, studies on the fate of IRT1 after fusion with PM in response to iron conditions are still limited. In this study, we found that K165 and K196 regulate the monoubiquitination of MxIRT1 (mUb-MxIRT1), which acts as a receptor delivering signals from HRM to downstream effectors such as clathrin to determine the fate of MxIRT1. Iron supply led MxIRT1 in the PM to monoubiquitin-dependent endocytosis which could be inhibited by endocytosis inhibitor TyrA23 or in the double site-directed mutant K165/K196R. Subsequently, the endocytosis pathway to the vacuole was inhibited by vacuolar protease inhibitor Leupeptin in excessive iron conditions and the inability of being able to respond to iron change, indicated by the protein accumulating in the PM, contributed to iron toxicity in K165/K196R transgenic Arabidopsis. With iron availability decreasing again, MxIRT1 could dock close to the PM waiting for to be recycled. Another monoubiquitination site, K26, was necessary for MxIRT1 Endoplasmic Reticulum (ER) export as site-directed mutant K26R lost the ability of PM targeting, and co-localized with the COPII subunit of the coat protein OsSec24. Therefore, after K26-directed ER export and iron-induced PM fusion, mUb-MxIRT1 determines subsequent vacuolar degradation or recycling to the PM via endocytosis for maintaining iron homeostasis.

A phosphorylation network controls the stability of alpha‐arrestins Aly1/Art6 and Aly2/Art3

Selective protein trafficking controls the repertoire of membrane proteins at the cell surface. This contingent of surface proteins regulates nutrient/metabolite balance and response to extracellular cues. Environmental changes trigger cellular signaling that drives transitions in the plasma membrane proteome. These alterations are achieved in no small part through regulation of the α‐arrestins, an emergent and powerful class of protein trafficking adaptors. The α‐arrestins are largely cytosolic proteins that are transiently recruited to membranes via binding to membrane protein motifs. α‐Arrestins bring with them a ubiquitin ligase, which stimulates ubiquitination and subsequent endocytosis of membrane proteins. Phosphorylation of α‐arrestins is key to regulating their trafficking function; dephosphorylated α‐arrestins are generally ‘active’ endocytic adaptors. While some kinases and phosphatases for α‐arrestins are known, the degree of α‐arrestins phosphorylation is staggering, with >40 phosphorylation sites identified for a single α‐arrestin, and suggests we still have much to learn about α‐arrestins regulation. We sought to determine what kinases and phosphates regulate paralogous α‐arrestins Aly1 and Aly2 using a genetic screen in yeast. Using an array of all known non‐essential kinase and phosphatase deletions, we determined which of these influenced α‐arrestin‐mediated resistance to rapamycin, an inhibitor of the TORC1 nutrient‐sensing kinase. We identified a large cohort of kinases and phosphates that influenced Aly‐dependent phenotypes, causing electrophoretic mobility changes and, in many cases, diminishing the abundance of these α‐arrestins. We focused our studies on the Sit4 protein phosphatase, a key regulator downstream of TORC1 signaling able to influence other α‐arrestins, identified in our screen. Strikingly, Aly1 and Aly2 were hyperphosphorylated and destabilized in the absence of Sit4, suggesting that excessive phosphorylation may promote degradation of these α‐arrestins. For Aly2, but not Aly1, degradation in the sit4∆ cells could be reversed by loss of the vacuolar protease Pep4, indicating that vacuolar degradation predominates for Aly2 under these conditions. Loss of the Npr1 kinase in sit4∆ cells restored Aly protein abundances and reversed the hyperphosphorylation, demonstrating that this kinase is responsible for the excess Aly phosphorylation in sit4∆ cells. This is a remarkable finding as typically Npr1 is considered inactive in sit4∆, however, we suggest that Npr1 is selectively active in the absence of Sit4, able to modify some substrates but not others. We define new features of TORC1 signaling in regulating α‐arrestin phosphorylation and stability.

Convergence Analysis of Rust Fungi and Anther Smuts Reveals Their Common Molecular Adaptation to a Phytoparasitic Lifestyle.

Convergent evolution between distantly related taxa often mirrors adaptation to similar environments. Rust fungi and anther smuts, which belong to different classes in Pucciniomycotina, have independently evolved a phytoparasitic lifestyle, representing an example of convergent evolution in the fungal kingdom. To investigate their adaptations and the genetic bases underlying their phytoparasitic lifestyles, we performed genome-wide convergence analysis of amino acid substitutions, evolutionary rates, and gene gains and losses. Convergent substitutions were detected in ATPeV0D and RP-S27Ae, two genes important for the generation of turgor pressure and ribosomal biosynthesis, respectively. A total of 51 positively selected genes were identified, including eight genes associated with translation and three genes related to the secretion pathway. In addition, rust fungi and anther smuts contained more proteins associated with oligopeptide transporters and vacuolar proteases than did other fungi. For rust fungi and anther smuts, these forms of convergence suggest four adaptive mechanisms for a phytoparasitic lifestyle: 1) reducing the metabolic demand for hyphal growth and penetration at the pre-penetration stage, 2) maintaining the efficiency of protein synthesis during colonization, 3) ensuring the normal secretion of rapidly evolving secreted proteins, and 4) improving the capacity for oligopeptide metabolism. Our results are the first to shed light on the genetic convergence mechanisms and molecular adaptation underlying phytoparasitic lifestyles in fungi.

Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules

BackgroundAffibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins.ResultsWe examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L.ConclusionThis study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

Determination of Putative Vacuolar Proteases, PEP4 and PRB1 in a Novel Yeast Expression Host Meyerozyma guilliermondii Strain SO Using Bioinformatics Tools

Meyerozyma guilliermondii strain SO, a newly isolated yeast species from spoilt orange, has been used as a host to express the recombinant proteins using methylotrophic yeast promoters. However, as a novel yeast expression system, the vacuolar proteases of this yeast have not been determined, which may have contributed to the low level of heterologous protein secretions. Thus, this study aimed to determine intra- and extracellular proteolytic activity and identify the putative vacuolar proteases using bioinformatics techniques. A clear zone was observed from the nutrient agar skimmed milk screening plate. Proteolytic activity of 117.30 U/ml and 75 U/ml were obtained after 72 h of cultivation for both extracellular and intracellular proteins, respectively. Next, the Hidden Markov model (HMM) was used to detect the presence of the vacuolar proteases (PEP4 and PRB1) from the strain SO proteome. Aspartyl protease (PEP4) with 97.55% identity to Meyerozyma sp. JA9 and a serine protease (PRB1) with 70.91% identity to Candida albicans were revealed. The homology with other yeast vacuolar proteases was confirmed via evolutionary analysis. PROSPER tool prediction of cleavage sites postulated that PEP4 and PRB1 might have caused proteolysis of heterologous proteins in strain SO. In conclusion, two putative vacuolar proteases (PEP4 and PRB1) were successfully identified in strain SO. Further characterization can be done to understand their specific properties, and their effects on heterologous protein expression can be conducted via genome editing.

Novel Nile Blue Analogue Stains Yeast Vacuolar Membrane, Endoplasmic Reticulum, and Lipid Droplets, Inducing Cell Death through Vacuole Membrane Permeabilization.

Phenoxazine derivatives such as Nile Blue analogues are assumed to be increasingly relevant in cell biology due to their fluorescence staining capabilities and antifungal and anticancer activities. However, the mechanisms underlying their effects remain poorly elucidated. Using S. cerevisiae as a eukaryotic model, we found that BaP1, a novel 5- and 9-N-substituted benzo[a]phenoxazine synthesized in our laboratory, when used in low concentrations, accumulates and stains the vacuolar membrane and the endoplasmic reticulum. In contrast, at higher concentrations, BaP1 stains lipid droplets and induces a regulated cell death process mediated by vacuolar membrane permeabilization. BaP1 also induced mitochondrial fragmentation and depolarization but did not lead to ROS accumulation, changes in intracellular Ca2+, or loss of plasma membrane integrity. Additionally, our results show that the cell death process is dependent on the vacuolar protease Pep4p and that the vacuole permeabilization results in its translocation from the vacuole to the cytosol. In addition, although nucleic acids are commonly described as targets of benzo[a]phenoxazines, we did not find any alterations at the DNA level. Our observations highlight BaP1 as a promising molecule for pharmacological application, using vacuole membrane permeabilization as a targeted approach.

Effect of high pressure on the proteolytic activity and autolysis of yeast Saccharomyces cerevisiae

This work explores the effect of High Pressure (HP) treatment on the proteolytic activity and autolytic capacity of Saccharomyces cerevisiae suspensions, based on yeast extract production. Cellular suspensions were treated at 200–600 MPa for 0–120 min and the activity of vacuolar proteases was measured during autolysis. Moreover, the autolytic capacity of yeast was determined based on the physicochemical characteristics of the produced yeast extract. At pressures of 200 and 400 MPa, proteolytic activity was enhanced up to 160%, after 40 and 10 min of HP treatment, respectively, leading to significantly accelerated autolysis, in combination with cellular permeabilization achieved with HP treatment. However, at 600 MPa, despite the greater cell permeabilization, proteolytic enzymes were gradually inactivated (total inactivation after 15 min of HP treatment), leading to inhibition of autolysis. These results highlight HP as an effective process to enhance endogenous proteolytic activity and thus accelerate yeast extract production.

Arabidopsis ADP-RIBOSYLATION FACTOR-A1s mediate tapetum-controlled pollen development.

Propagation of angiosperms mostly relies on sexual reproduction, in which gametophytic development is a pre-requisite. Male gametophytic development requires both gametophytic and sporophytic factors, most importantly early secretion and late programmed cell death of the tapetum. In addition to transcriptional factors, proteins at endomembrane compartments, such as receptor-like kinases and vacuolar proteases, control tapetal function. The cellular machinery that regulates their distribution is beginning to be revealed. We report here that ADP-RIBOSYLATION FACTOR-A1s (ArfA1s) are critical for tapetum-controlled pollen development. All six ArfA1s in the Arabidopsis genome are expressed during anther development, among which ArfA1b is specific to the tapetum and developing microspores. Although the ArfA1b loss-of-function mutant showed no pollen defects, probably due to redundancy, interference with ArfA1s by a dominant negative approach in the tapetum resulted in tapetal dysfunction and pollen abortion. We further showed that all six ArfA1s are associated with the Golgi and the trans-Golgi network/early endosome, suggesting that they have roles in regulating post-Golgi trafficking to the plasma membrane or to vacuoles. Indeed, we demonstrated that the expression of ArfA1bDN interfered with the targeting of proteins critical for tapetal development. The results presented here demonstrate a key role of ArfA1s in tapetum-controlled pollen development by mediating protein targeting through post-Golgi trafficking routes.

Lipid Droplets and Their Autophagic Turnover via the Raft-Like Vacuolar Microdomains.

Although once perceived as inert structures that merely serve for lipid storage, lipid droplets (LDs) have proven to be the dynamic organelles that hold many cellular functions. The LDs’ basic structure of a hydrophobic core consisting of neutral lipids and enclosed in a phospholipid monolayer allows for quick lipid accessibility for intracellular energy and membrane production. Whereas formed at the peripheral and perinuclear endoplasmic reticulum, LDs are degraded either in the cytosol by lipolysis or in the vacuoles/lysosomes by autophagy. Autophagy is a regulated breakdown of dysfunctional, damaged, or surplus cellular components. The selective autophagy of LDs is called lipophagy. Here, we review LDs and their degradation by lipophagy in yeast, which proceeds via the micrometer-scale raft-like lipid domains in the vacuolar membrane. These vacuolar microdomains form during nutrient deprivation and facilitate internalization of LDs via the vacuolar membrane invagination and scission. The resultant intra-vacuolar autophagic bodies with LDs inside are broken down by vacuolar lipases and proteases. This type of lipophagy is called microlipophagy as it resembles microautophagy, the type of autophagy when substrates are sequestered right at the surface of a lytic compartment. Yeast microlipophagy via the raft-like vacuolar microdomains is a great model system to study the role of lipid domains in microautophagic pathways.

Post-ER degradation of misfolded GPI-anchored proteins is linked with microautophagy

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are membrane-conjugated cell-surface proteins with diverse structural, developmental, and signaling functions and clinical relevance. Typically, after biosynthesis and attachment to the preassembled GPI anchor, GPI-APs rapidly leave the endoplasmic reticulum (ER) and rely on post-ER quality control. Terminally misfolded GPI-APs end up inside the vacuole/lysosome for degradation, but their trafficking itinerary to this organelle and the processes linked to their uptake by the vacuole/lysosome remain uncharacterized. In a yeast mutant that is lacking Pep4, a key vacuolar protease, several misfolded model GPI-APs accumulated in the vacuolar membrane. In the same mutant, macroautophagy and the multi-vesicular body (MVB) pathway were intact, hinting at a hitherto-unknown trafficking pathway for the degradation of misfolded GPI-APs. To unravel it, we used a genome-wide screen coupled to high-throughput fluorescence microscopy and followed the fate of the misfolded GPI-AP: Gas1∗. We found that components of the early secretory and endocytic pathways are involved in its targeting to the vacuole and that vacuolar transporter chaperones (VTCs), with roles in microautophagy, negatively affect the vacuolar uptake of Gas1∗. In support, we demonstrate that Gas1∗ internalizes from vacuolar membranes into membrane-bound intravacuolar vesicles prior to degradation. Our data link post-ER degradation with microautophagy.

Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes — these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.