BnXTH1 regulates cadmium tolerance by modulating vacuolar compartmentalization and the cadmium binding capacity of cell walls in ramie (Boehmeria nivea).

Xyloglucan endotransglucosylase/hydrolases (XTH) are cell wall-modifying enzymes important in plant response to abiotic stress. However, the role of XTH in cadmium (Cd) tolerance in ramie remains largely unknown. Here, we identified and cloned BnXTH1, a member of the XTH family, in response to Cd stress in ramie. The BnXTH1 promoter (BnXTH1p) demonstrated that MeJA induces the response of BnXTH1p to Cd stress. Moreover, overexpressing BnXTH1 in Boehmeria nivea increased Cd tolerance by significantly increasing the Cd content in the cell wall and decreasing Cd inside ramie cells. Cadmium stress induced BnXTH1-expression and consequently increased xyloglucan endotransglucosylase (XET) activity, leading to high xyloglucan contents and increased hemicellulose contents in ramie. The elevated hemicellulose content increased Cd chelation onto the cell walls and reduced the level of intracellular Cd. Interestingly, overexpressing BnXTH1 significantly increased the content of Cd in vacuoles of ramie and vacuolar compartmentalization genes. Altogether, these results evidence that Cd stress induced MeJA accumulation in ramie, thus, activating BnXTH1 expression and increasing the content of xyloglucan to enhance the hemicellulose binding capacity and increase Cd chelation onto cell walls. BnXTH1 also enhances the vacuolar Cd compartmentalization and reduces the level of Cd entering the organelles and soluble solution.

Sit4 Genetically Interacts with Vps27 to Regulate Mitochondrial Function and Lifespan in Saccharomyces cerevisiae.

The Sit4 protein phosphatase plays a key role in orchestrating various cellular processes essential for maintaining cell viability during aging. We have previously shown that SIT4 deletion promotes vacuolar acidification, mitochondrial derepression, and oxidative stress resistance, increasing yeast chronological lifespan. In this study, we performed a proteomic analysis of isolated vacuoles and yeast genetic interaction analysis to unravel how Sit4 influences vacuolar and mitochondrial function. By employing high-resolution mass spectrometry, we show that sit4Δ vacuolar membranes were enriched in Vps27 and Hse1, two proteins that are part of the endosomal sorting complex required for transport-0. In addition, SIT4 exhibited a negative genetic interaction with VPS27, as sit4∆vps27∆ double mutants had a shortened lifespan compared to sit4∆ and vps27∆ single mutants. Our results also show that Vps27 did not increase sit4∆ lifespan by improving protein trafficking or vacuolar sorting pathways. However, Vps27 was critical for iron homeostasis and mitochondrial function in sit4∆ cells, as sit4∆vps27∆ double mutants exhibited high iron levels and impaired mitochondrial respiration. These findings show, for the first time, cross-talk between Sit4 and Vps27, providing new insights into the mechanisms governing chronological lifespan.

Post-translational targeting of Rab35 by the effector IcsB of Shigella determines intracellular bacterial niche formation.

Escape from the bacterial-containing vacuole (BCV) is a key step of Shigella host cell invasion. Rab GTPases subverted to in situ-formed macropinosomes in the vicinity of the BCV have been shown to promote its rupture. The involvement of the BCV itself has remained unclear. We demonstrate that Rab35 is non-canonically entrapped at the BCV. Stimulated emission depletion imaging localizes Rab35 directly on the BCV membranes before vacuolar rupture. The bacterial effector IcsB, a lysine Nε-fatty acylase, is a key regulator of Rab35-BCV recruitment, and we show post-translational acylation of Rab35 by IcsB in its polybasic region. While Rab35 and IcsB are dispensable for the first step of BCV breakage, they are needed for the unwrapping of damaged BCV remnants from Shigella. This provides a framework for understanding Shigella invasion implicating re-localization of a Rab GTPase via its bacteria-dependent post-translational modification to support the mechanical unpeeling of the BCV.

The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling.

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.

Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion.

Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.

iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse.

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, this mechanism is incomplete. Here, using automated spatially targeted optical micro proteomics we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.

Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

Membrane contact sites regulate vacuolar fission via sphingolipid metabolism.

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus-vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Autophagy related genes mediated mitophagy in yeast, mammals and higher plants.

Autophagy is classified into macro-autophagy and micro-autophagy. Two major types of autophagy in the complex eukaryotic organism are microautophagy and macroautophagy. During microautophagy, cytoplasmic components that need to be degraded are taken up by lysosomes in animals and by vacuole in yeast and plants via the invagination of tonoplast. While macroautophagy is initiated after the formation of a cup-shaped membrane structure, a phagophore develops at cargo that grows in size and is sealed by double-membrane vesicles to form autophagosome; a generalized mechanism for degradation of the organelle. Autophagic removal of damaged mitochondria is a conserved cellular process to maintain a healthy mitochondrion called Mitophagy. In plants and animals, mitophagy has crucial roles in stress responses, senescence, development, and programmed cell death. Mitophagy appears in mammals, fungi, and plants but many genes that controlled mitophagy are absent from plants. Numerous studies have been conducted by using ATG mutants for the identification of functional roles of Autophagy Related Genes (ATG) required during the autophagy process at various steps like; auto phagosome formation, ATG protein recruitment, etc. The role of more than 25 ATG genes in mitophagy has been discussed in this review paper. The main parameters, reviewed and summarized in this review paper, are the name of species, common name, function, domain, deletion, induction, and localization of these autophagy-related genes in the cell. This review will facilitate the students, researchers, and academics for their further research insights.

Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins secreted from dense granule organelles to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to Δgra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions, we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant "bubble vacuole" morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δgra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections. IMPORTANCE: Toxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the PVM, which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM's permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47 or those with a histidine mutation have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against toxoplasmosis.

Palmitoylation of vacuole membrane protein 1 promotes small extracellular vesicle secretion via interaction with ALIX and influences intercellular communication.

BACKGROUND: Small extracellular vesicles (EVs), exemplified by exosomes, mediate intercellular communication by transporting proteins, mRNAs, and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However, whether palmitoylation regulates small EV secretion, remains largely unexplored. METHODS: Vacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated, identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1, while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication, SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo, mouse model was established by intraperitoneal injection with palmitoylation inhibitor, 2-bromopalmitate (2BP) for 3 months. RESULTS: VMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically, palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore, inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX, an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication, we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance, increased apoptosis, and decreased proliferation rate of SSCLCs. In vivo, intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes, which were closely associated with the level of spermatogenic cell apoptosis and proliferation. CONCLUSIONS: Our study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells, and demonstrated its pivotal role in intercellular communication and SSC niche.

Altered vacuole membrane protein 1 (VMP1) expression is associated with increased NLRP3 inflammasome activation and mitochondrial dysfunction.

BACKGROUND: Altered expression of vacuole membrane protein 1 (VMP1) has recently been observed in the context of multiple sclerosis and Parkinson's disease (PD). However, how changes in VMP1 expression may impact pathogenesis has not been explored. OBJECTIVE: This study aimed to characterize how altered VMP1 expression affects NLRP3 inflammasome activation and mitochondrial function. METHODS: VMP1 expression was depleted in a monocytic cell line using CRISPR-Cas9. The effect of VMP1 on NLRP3 inflammasome activation was examined by stimulating cells with LPS and ATP or α-synuclein fibrils. Inflammasome activation was determined by caspase-1 activation using both a FLICA assay and a biosensor as well as by the release of proinflammatory molecules measured by ELISA. RNA-sequencing was utilized to define global gene expression changes resulting from VMP1 deletion. SERCA activity and mitochondrial function were investigated using various fluorescence microscopy-based approaches including a novel method that assesses the function of individual mitochondria in a cell. RESULTS: Here, we report that genetic deletion of VMP1 from a monocytic cell line resulted in increased NLRP3 inflammasome activation and release of proinflammatory molecules. Examination of the VMP1-dependent changes in these cells revealed that VMP1 deficiency led to decreased SERCA activity and increased intracellular [Ca2+]. We also observed calcium overload in mitochondria in VMP1 depleted cells, which was associated with mitochondrial dysfunction and release of mitochondrial DNA into the cytoplasm and the extracellular environment. CONCLUSIONS: Collectively, these studies reveal VMP1 as a negative regulator of inflammatory responses, and we postulate that decreased expression of VMP1 can aggravate the inflammatory sequelae associated with neurodegenerative diseases like PD.

Microtubules provide force to promote membrane uncoating in vacuolar escape for a cyto-invasive bacterial pathogen.

Intracellular bacterial pathogens gain entry to mammalian cells inside a vacuole derived from the host membrane. Some of them escape the bacteria-containing vacuole (BCV) and colonize the cytosol. Bacteria replicating within BCVs coopt the microtubule network to position it within infected cells, whereas the role of microtubules for cyto-invasive pathogens remains obscure. Here, we show that the microtubule motor cytoplasmic dynein-1 and specific activating adaptors are hijacked by the enterobacterium Shigella flexneri. These host proteins were found on infection-associated macropinosomes (IAMs) formed during Shigella internalization. We identified Rab8 and Rab13 as mediators of dynein recruitment and discovered that the Shigella effector protein IpaH7.8 promotes Rab13 retention on moving BCV membrane remnants, thereby facilitating membrane uncoating of the Shigella-containing vacuole. Moreover, the efficient unpeeling of BCV remnants contributes to a successful intercellular spread. Taken together, our work demonstrates how a bacterial pathogen subverts the intracellular transport machinery to secure a cytosolic niche.

LDO proteins and Vac8 form a vacuole-lipid droplet contact site to enable starvation-induced lipophagy in yeast.

Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via lipophagy, the en bloc uptake of LDs into the vacuole. Here, we show that LDs dock to the vacuolar membrane via a contact site that is required for lipophagy in yeast. The LD-localized LDO proteins carry an intrinsically disordered region that directly binds vacuolar Vac8 to form vCLIP, the vacuolar-LD contact site. Nutrient limitation drives vCLIP formation, and its inactivation blocks lipophagy, resulting in impaired caloric restriction-induced longevity. We establish a functional link between lipophagy and microautophagy of the nucleus, both requiring Vac8 to form respective contact sites upon metabolic stress. In sum, we identify the tethering machinery of vCLIP and find that Vac8 provides a platform for multiple and competing contact sites associated with autophagy.

A metabolically controlled contact site between vacuoles and lipid droplets in yeast.

The lipid droplet (LD) organization proteins Ldo16 and Ldo45 affect multiple aspects of LD biology in yeast. They are linked to the LD biogenesis machinery seipin, and their loss causes defects in LD positioning, protein targeting, and breakdown. However, their molecular roles remained enigmatic. Here, we report that Ldo16/45 form a tether complex with Vac8 to create vacuole lipid droplet (vCLIP) contact sites, which can form in the absence of seipin. The phosphatidylinositol transfer protein (PITP) Pdr16 is a further vCLIP-resident recruited specifically by Ldo45. While only an LD subpopulation is engaged in vCLIPs at glucose-replete conditions, nutrient deprivation results in vCLIP expansion, and vCLIP defects impair lipophagy upon prolonged starvation. In summary, Ldo16/45 are multifunctional proteins that control the formation of a metabolically regulated contact site. Our studies suggest a link between LD biogenesis and breakdown and contribute to a deeper understanding of how lipid homeostasis is maintained during metabolic challenges.

Legionella pneumophila usurps host cell lipids for vacuole expansion and bacterial growth.

Vacuolar pathogens reside in membrane-bound compartments within host cells. Maintaining the integrity of this compartment is paramount to bacterial survival and replication as it protects against certain host surveillance mechanisms that function to eradicate invading pathogens. Preserving this compartment during bacterial replication requires expansion of the vacuole membrane to accommodate the increasing number of bacteria, and yet, how this is accomplished remains largely unknown. Here, we show that the vacuolar pathogen Legionella pneumophila exploits multiple sources of host cell fatty acids, including inducing host cell fatty acid scavenging pathways, in order to promote expansion of the replication vacuole and bacteria growth. Conversely, when exogenous lipids are limited, the decrease in host lipid availability restricts expansion of the replication vacuole membrane, resulting in a higher density of bacteria within the vacuole. Modifying the architecture of the vacuole prioritizes bacterial growth by allowing the greatest number of bacteria to remain protected by the vacuole membrane despite limited resources for its expansion. However, this trade-off is not without risk, as it can lead to vacuole destabilization, which is detrimental to the pathogen. However, when host lipid resources become extremely scarce, for example by inhibiting host lipid scavenging, de novo biosynthetic pathways, and/or diverting host fatty acids to storage compartments, bacterial replication becomes severely impaired, indicating that host cell fatty acid availability also directly regulates L. pneumophila growth. Collectively, these data demonstrate dual roles for host cell fatty acids in replication vacuole expansion and bacterial proliferation, revealing the central functions for these molecules and their metabolic pathways in L. pneumophila pathogenesis.

Toxoplasma and Plasmodium associate with host Arfs during infection.

The apicomplexans Toxoplasma gondii and Plasmodium are intracellular parasites that reside within a host-derived compartment termed the parasitophorous vacuole (PV). During infection, the parasites must acquire critical host resources and transport them across their PV for development. However, the mechanism by which host resources are trafficked to and across the PV remains uncertain. Here, we investigated host ADP ribosylation factors (Arfs), a class of proteins involved in vesicular trafficking that may be exploited by T. gondii and Plasmodium berghei for nutrient acquisition. Using overexpressed Arf proteins coupled with immunofluorescence microscopy, we found that all Arfs were internalized into the T. gondii PV, with most vacuoles containing at least one punctum of Arf protein by the end of the lytic cycle. We further characterized Arf1, the most abundant Arf inside the T. gondii PV, and observed that active recycling between its GDP/GTP-bound state influenced Arf1 internalization independent of host guanine nucleotide exchange factors (GEFs). In addition, Arf1 colocalized with vesicle coat complexes and exogenous sphingolipids, suggesting a role in nutrient acquisition. While Arf1 and Arf4 were not observed inside the PV during P. berghei infection, our gene depletion studies showed that liver stage development and survival depended on the expression of Arf4 and the host GEF, GBF1. Collectively, these observations indicate that apicomplexans use distinct mechanisms to subvert the host vesicular trafficking network and efficiently replicate. The findings also pave the way for future studies to identify parasite proteins critical to host vesicle recruitment and the components of vesicle cargo. IMPORTANCE: The parasites Toxoplasma gondii and Plasmodium live complex intracellular lifestyles where they must acquire essential host nutrients while avoiding recognition. Although previous work has sought to identify the specific nutrients scavenged by apicomplexans, the mechanisms by which host materials are transported to and across the parasite vacuole membrane are largely unknown. Here, we examined members of the host vesicular trafficking network to identify specific pathways subverted by T. gondii and Plasmodium berghei. Our results indicate that T. gondii selectively internalizes host Arfs, a class of proteins involved in intracellular trafficking. For P. berghei, host Arfs were restricted by the parasite's vacuole membrane, but proteins involved in vesicular trafficking were identified as essential for liver stage development. A greater exploration into how and why apicomplexans subvert host vesicular trafficking could help identify targets for host-directed therapeutics.

Transforming yeast into a facultative photoheterotroph via expression of vacuolar rhodopsin.

Phototrophic metabolism, the capture of light for energy, was a pivotal biological innovation that greatly increased the total energy available to the biosphere. Chlorophyll-based photosynthesis is the most familiar phototrophic metabolism, but retinal-based microbial rhodopsins transduce nearly as much light energy as chlorophyll does,1 via a simpler mechanism, and are found in far more taxonomic groups. Although this system has apparently spread widely via horizontal gene transfer,2,3,4 little is known about how rhodopsin genes (with phylogenetic origins within prokaryotes5,6) are horizontally acquired by eukaryotic cells with complex internal membrane architectures or the conditions under which they provide a fitness advantage. To address this knowledge gap, we sought to determine whether Saccharomyces cerevisiae, a heterotrophic yeast with no known evolutionary history of phototrophy, can function as a facultative photoheterotroph after acquiring a single rhodopsin gene. We inserted a rhodopsin gene from Ustilago maydis,7 which encodes a proton pump localized to the vacuole, an organelle normally acidified via a V-type rotary ATPase, allowing the rhodopsin to supplement heterotrophic metabolism. Probes of the physiology of modified cells show that they can deacidify the cytoplasm using light energy, demonstrating the ability of rhodopsins to ameliorate the effects of starvation and quiescence. Further, we show that yeast-bearing rhodopsins gain a selective advantage when illuminated, proliferating more rapidly than their non-phototrophic ancestor or rhodopsin-bearing yeast cultured in the dark. These results underscore the ease with which rhodopsins may be horizontally transferred even in eukaryotes, providing novel biological function without first requiring evolutionary optimization.

Toxoplasma protein export and effector function.

Toxoplasma gondii is a single-celled eukaryotic parasite with a considerable host range that must invade the cells of warm-blooded hosts to survive and replicate. The challenges and opportunities that such a strategy represent have been met by the evolution of effectors that are delivered into host cells, counter host defences and co-opt host cell functions for their own purposes. These effectors are delivered in two waves using distinct machinery for each. In this Review, we focus on understanding the architecture of these protein-export systems and how their protein cargo is recognized and selected. We discuss the recent findings on the role that host manipulation has in latent Toxoplasma infections. We also discuss how these recent findings compare to protein export in the related Plasmodium spp. (the causative agent of malaria) and how this can inform our understanding of host manipulation in the larger Apicomplexa phylum and its evolution.

Methionine sulfoxide reductase 2 regulates Cvt autophagic pathway by altering the stability of Atg19 and Ape1 in Saccharomyces cerevisiae.

The reversible oxidation of methionine plays a crucial role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can destabilize and abrogate their function. The highly conserved methionine sulfoxide reductases protect proteins from oxidative damage by reducing their oxidized methionines, thus restoring their stability and function. Deletion or mutation in conserved methionine sulfoxide reductases leads to aging and several human neurological disorders and also reduces yeast growth on nonfermentable carbon sources. Despite their importance in human health, limited information about their physiological substrates in humans and yeast is available. For the first time, we show that Mxr2 interacts in vivo with two core proteins of the cytoplasm to vacuole targeting (Cvt) autophagy pathway, Atg19, and Ape1 in Saccharomyces cerevisiae. Deletion of MXR2 induces instability and early turnover of immature Ape1 and Atg19 proteins and reduces the leucine aminopeptidase activity of Ape1 without affecting the maturation process of Ape1. Additonally, Mxr2 interacts with the immature Ape1, dependent on Met17 present within the propeptide of Ape1 as a single substitution mutation of Met17 to Leu abolishes this interaction. Importantly, Ape1 M17L mutant protein resists oxidative stress-induced degradation in WT and mxr2Δ cells. By identifying Atg19 and Ape1 as cytosolic substrates of Mxr2, our study maps the hitherto unexplored connection between Mxr2 and the Cvt autophagy pathway and sheds light on Mxr2-dependent oxidative regulation of the Cvt pathway.