Towards solving the mystery of peroxisomal matrix protein import.

Peroxisomes are vital metabolic organelles that import their lumenal (matrix) enzymes from the cytosol using mobile receptors. Surprisingly, the receptors can even import folded proteins, but the underlying mechanism has been a mystery. Recent results reveal how import receptors shuttle cargo into peroxisomes. The cargo-bound receptors move from the cytosol across the peroxisomal membrane completely into the matrix by a mechanism that resembles transport through the nuclear pore. The receptors then return to the cytosol through a separate retrotranslocation channel, leaving the cargo inside the organelle. This cycle concentrates imported proteins within peroxisomes, and the energy for cargo import is supplied by receptor export. Peroxisomal protein import thus fundamentally differs from other previously known mechanisms for translocating proteins across membranes.

Cell-free reconstitution of peroxisomal matrix protein import using Xenopus egg extract.

Peroxisomes are vital metabolic organelles whose matrix enzymes are imported from the cytosol in a folded state by the soluble receptor PEX5. The import mechanism has been challenging to decipher because of the lack of suitable in vitro systems. Here, we present a protocol for reconstituting matrix protein import using Xenopus egg extract. We describe how extract is prepared, how to replace endogenous PEX5 with recombinant versions, and how to perform and interpret a peroxisomal import reaction using a fluorescent cargo. For complete details on the use and execution of this protocol, please refer to Skowyra and Rapoport (2022).1.

Protein import into peroxisomes occurs through a nuclear pore-like phase.

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal proteins are imported from the cytosol in a folded state by the soluble receptor PEX5. How folded cargo crosses the membrane is unknown. Here, we show that peroxisomal import is similar to nuclear transport. The peroxisomal membrane protein PEX13 contains a conserved tyrosine (Y)- and glycine (G)-rich YG domain, which forms a selective phase resembling that formed by phenylalanine-glycine (FG) repeats within nuclear pores. PEX13 resides in the membrane in two orientations that oligomerize and suspend the YG meshwork within the lipid bilayer. Purified YG domains form hydrogels into which PEX5 selectively partitions, by using conserved aromatic amino acid motifs, bringing cargo along. The YG meshwork thus forms an aqueous conduit through which PEX5 delivers folded proteins into peroxisomes.

Structure and function of the peroxisomal ubiquitin ligase complex.

Peroxisomes are membrane-bounded organelles that exist in most eukaryotic cells and are involved in the oxidation of fatty acids and the destruction of reactive oxygen species. Depending on the organism, they house additional metabolic reactions that range from glycolysis in parasitic protozoa to the production of ether lipids in animals and antibiotics in fungi. The importance of peroxisomes for human health is revealed by various disorders - notably the Zellweger spectrum - that are caused by defects in peroxisome biogenesis and are often fatal. Most peroxisomal metabolic enzymes reside in the lumen, but are synthesized in the cytosol and imported into the organelle by mobile receptors. The receptors accompany cargo all the way into the lumen and must return to the cytosol to start a new import cycle. Recycling requires receptor monoubiquitination by a membrane-embedded ubiquitin ligase complex composed of three RING finger (RF) domain-containing proteins: PEX2, PEX10, and PEX12. A recent cryo-electron microscopy (cryo-EM) structure of the complex reveals its function as a retro-translocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that assemble into an open channel. The N terminus of a receptor likely inserts into the pore from the lumenal side, and is then monoubiquitinated by one of the RFs to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitinated by the concerted action of the other two RFs and ultimately degraded. The new data provide mechanistic insight into a crucial step of peroxisomal protein import.

PEX5 translocation into and out of peroxisomes drives matrix protein import.

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal enzymes are imported from the cytosol by the receptor PEX5, which interacts with a docking complex in the peroxisomal membrane and then returns to the cytosol after monoubiquitination by a membrane-embedded ubiquitin ligase. The mechanism by which PEX5 shuttles between cytosol and peroxisomes and releases cargo inside the lumen is unclear. Here, we use Xenopus egg extract to demonstrate that PEX5 accompanies cargo completely into the lumen, utilizing WxxxF/Y motifs near its N terminus that bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix that binds to the lumenal side of the ubiquitin ligase. The N terminus then emerges in the cytosol for monoubiquitination. Finally, PEX5 is extracted from the lumen, resulting in the unfolding of the receptor and cargo release. Our results reveal the unique mechanism by which PEX5 ferries proteins into peroxisomes.

Mycobacterium tuberculosis Type VII Secretion System Effectors Differentially Impact the ESCRT Endomembrane Damage Response.

Intracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case of Mycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and that M. tuberculosis could impair this host response. Indeed, we found that ESCRT-III proteins were recruited to M. tuberculosis phagosomes in an ESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context of M. tuberculosis infection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity.IMPORTANCEMycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.

Triggered recruitment of ESCRT machinery promotes endolysosomal repair.

Endolysosomes can be damaged by diverse materials. Terminally damaged compartments are degraded by lysophagy, but pathways that repair salvageable organelles are poorly understood. Here we found that the endosomal sorting complex required for transport (ESCRT) machinery, known to mediate budding and fission on endolysosomes, also plays an essential role in their repair. ESCRTs were rapidly recruited to acutely injured endolysosomes through a pathway requiring calcium and ESCRT-activating factors that was independent of lysophagy. We used live-cell imaging to demonstrate that ESCRTs responded to small perforations in endolysosomal membranes and enabled compartments to recover from limited damage. Silica crystals that disrupted endolysosomes also triggered ESCRT recruitment. ESCRTs thus provide a defense against endolysosomal damage likely to be relevant in physiological and pathological contexts.

Structure and membrane remodeling activity of ESCRT-III helical polymers.

The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises "open" CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of "closed" IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.

Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation.

Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus-its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and "foreman" TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes.

Cryptococcus neoformans dual GDP-mannose transporters and their role in biology and virulence.

Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.

Identification of the galactosyltransferase of Cryptococcus neoformans involved in the biosynthesis of basidiomycete-type glycosylinositolphosphoceramide.

The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by ß1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdom.

Unusual galactofuranose modification of a capsule polysaccharide in the pathogenic yeast Cryptococcus neoformans.

Galactofuranose (Galf) is the five-membered ring form of galactose. Although it is absent from mammalian glycans, it occurs as a structural and antigenic component of important cell surface molecules in a variety of microbes, ranging from bacteria to parasites and fungi. One such organism is Cryptococcus neoformans, a pathogenic yeast that causes lethal meningoencephalitis in immunocompromised individuals, particularly AIDS patients. C. neoformans is unique among fungal pathogens in bearing a complex polysaccharide capsule, a critical virulence factor reported to include Galf. Notably, how Galf modification contributes to the structure and function of the cryptococcal capsule is not known. We have determined that Galf is ß1,2-linked to an unusual tetrasubstituted galactopyranose of the glucuronoxylomannogalactan (GXMGal) capsule polysaccharide. This discovery fills a longstanding gap in our understanding of a major polymer of the cryptococcal capsule. We also engineered a C. neoformans strain that lacks UDP-galactopyranose mutase; this enzyme forms UDP-Galf, the nucleotide sugar donor required for Galf addition. Mutase activity was required for the incorporation of Galf into glucuronoxylomannogalactan but was dispensable for vegetative growth, cell integrity, and virulence in a mouse model.

RNA interference in Cryptococcus neoformans.

RNA interference (RNAi) is an experimental technique used to suppress individual gene expression in eukaryotic cells in a sequence-dependent manner. The process relies on double-stranded RNA (dsRNA) to target complementary messenger RNA for degradation. Here, we describe two plasmid-based strategies we have developed for RNAi in Cryptococcus neoformans. The pFrame vector utilizes the ACT1 promoter to enable the constitutive synthesis of hairpin RNA (hpRNA), the stem of which constitutes the dsRNA trigger. The pIBB103 vector relies on convergent, inducible GAL7 promoters to independently drive the synthesis of the sense and antisense strands of the interfering sequence; these strands anneal to form the initiating dsRNA molecule. Both vectors are designed to co-silence a "sentinel" gene with an easily scored phenotype to help identify clones in which RNAi is most effective. We provide guidelines for selecting a suitable interfering sequence to trigger RNAi in C. neoformans and describe the steps for subcloning into either vector, transforming C. neoformans by electroporation, screening clones for RNAi-related phenotypes, and evaluating the efficacy and specificity of gene silencing by RNAi.

Toward an integrated model of capsule regulation in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation.

Emerging themes in cryptococcal capsule synthesis.

Cryptococcus neoformans, a basidiomycete yeast and opportunistic pathogen, expends significant biosynthetic effort on construction of a polysaccharide capsule with a radius that may be many times that of the cell. Beyond posing a stimulating challenge in terms of defining biosynthetic pathways, the capsule is required for this yeast to cause fatal disease. This combination has focused the attention of researchers on this system. Here we briefly review two aspects of the rapidly advancing field of capsule synthesis: the extensive variation that occurs in capsule polymers and the regulation of capsule biosynthesis.

A xylosylphosphotransferase of Cryptococcus neoformans acts in protein O-glycan synthesis.

Cryptococcal meningoencephalitis is an AIDS-defining illness caused by the opportunistic pathogen Cryptococcus neoformans. This organism possesses an elaborate polysaccharide capsule that is unique among pathogenic fungi, and the glycobiology of C. neoformans has been a focus of research in the field. The capsule and other cellular glycans and glycoconjugates have been described, but the machinery responsible for their synthesis remains largely unexplored. We recently discovered Xpt1p, an enzyme with the unexpected activity of generating a xylose-phosphate-mannose linkage. We now demonstrate that this novel activity is conserved throughout the C. neoformans species complex, localized to the Golgi apparatus, and functions in the O-glycosylation of proteins. We also present the first survey of O-glycans from C. neoformans.