PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway.

Little is known about eukaryotic chemorepulsion. The enzymes Phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3, and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion.

Dictyostelium discoideum cells retain nutrients when the cells are about to outgrow their food source.

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually outgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to outgrow their food supply and starve, causing the D. discoideum cells to inhibit their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases plasma membrane recycling and apparent cell membrane fluidity, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1 and the inositol hexakisphosphate kinase I6kA. PolyP alters protein contents in detergent-insoluble crude cytoskeletons, but does not significantly affect random cell motility, cell speed or F-actin levels. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve. This article has an associated First Person interview with the first author of the paper.

Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells.

Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as Mycobacterium tuberculosis accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. Escherichia coli K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by Dictyostelium discoideum or human macrophages. In contrast, Mycobacterium smegmatis and Mycobacterium tuberculosis accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by D. discoideum or macrophages. Adding extracellular polyphosphate increased E. coli survival after phagocytosis by D. discoideum and macrophages. Reducing expression of polyphosphate kinase 1 in M. smegmatis reduced extracellular polyphosphate and reduced survival in D. discoideum and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of D. discoideum and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed M. smegmatis or M. tuberculosisD. discoideum cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed E. coli and M. smegmatis Polyphosphate inhibited phagosome acidification and lysosome activity in D. discoideum and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation.

Starvation Induces Extracellular Accumulation of Polyphosphate in Dictyostelium discoideum to Inhibit Macropinocytosis, Phagocytosis, and Exocytosis.

Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity; this effect requires GrlD and Ppk1, but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, and to both decrease energy expenditures and retain nutrients.

Gallein potentiates isoniazid's ability to suppress Mycobacterium tuberculosis growth.

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug tolerance. Increased intracellular polyphosphate (polyP) in Mtb enhances tolerance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb's ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB.

Proteomic Analysis of Dictyostelium discoideum by Mass Spectrometry.

The large-scale proteomic analysis of Dictyostelium discoideum has contributed to our understanding of intracellular as well as secreted proteins in this versatile model eukaryote. Mass spectrometry-based proteomic analysis is a robust, sensitive, and rapid analytical method for identification and characterization of proteins extracted from tissues, cells, cell fractions, or pull-down assays. The availability of core facilities which make proteomics inexpensive and easy to do has facilitated a wide range of research projects. In this chapter, we present a simple standard methodology to extract proteins and prepare samples from D. discoideum for mass spectrometry and methods to analyze the identified proteins.

Gallein and isoniazid act synergistically to attenuate Mycobacterium tuberculosis growth in human macrophages.

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug resistance. Increased intracellular polyphosphate (polyP) in Mtb enhances resistance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb's ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB.

PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway.

The directed movement of eukaryotic cells is crucial for processes such as embryogenesis and immune cell trafficking. The enzyme Phosphatase and tensin homolog (PTEN) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. Dictyostelium discoideum cells require both PTEN and the PTEN-like phosphatase CnrN to locally inhibit Ras activation to induce biased movement of cells away from the secreted chemorepellent protein AprA. Both PTEN and CnrN decrease basal levels of PI(3,4,5)P 3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P 2 levels, decrease PI(3,4,5)P 3 levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. AprA causes PTEN, similar to CnrN, to localize to the side of the cell towards AprA in an AprA gradient. However, PTEN and CnrN also have distinct roles in some signaling pathways. PTEN, but not CnrN, decreases basal levels of PI(4,5)P 2 , AprA requires PTEN, but not CnrN, to induce cell roundness, and CnrN and PTEN have different effects on the number of filopods and pseudopods, and the sizes of filopods. Together, our results suggest that CnrN and PTEN play unique roles in D. discoideum signaling pathways, and possibly dephosphorylate PI(3,4,5)P 3 in different membrane domains, to mediate chemorepulsion away from AprA.

Starvation induces extracellular accumulation of polyphosphate in Dictyostelium discoideum to inhibit macropinocytosis, phagocytosis, and exocytosis.

Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity, and this effect requires GrlD and Ppk1 but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, to both decrease energy expenditures and retain nutrients.

Molecular simulation, vibrational spectroscopy and global reactivity descriptors of pseudoephedrine molecule in different phases and states.

The ground state molecular energy, vibrational frequencies and HOMO-LUMO analysis of the title compound have been calculated with density functional theory in the B3LYP/6-311 + G (d,p) basis set using Gaussian 09 W software. The FT-IR spectrum of pseudoephedrine has been computed in the gas phase and in the presence of solvent water both in neutral and anionic structures. The TED assignments of the vibrational spectra have been assigned in the selected intense region. On isotopic substitution of carbon atoms, the shifting of frequencies is distinctly observed. The reported values and HOMO-LUMO mappings reveal the possibility of different charge transfers occurring within the molecule. A MEP map is depicted and the Mulliken atomic charge is also calculated. The UV-Vis spectra have been illustrated and explained from the frontier molecular orbitals using a TD-DFT approach.

Polyphosphate uses mTOR, pyrophosphate, and Rho GTPase components to potentiate bacterial survival in Dictyostelium.

IMPORTANCE: Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic Mycobacterium species secrete polyP, and the polyP is necessary for the bacteria to prevent their killing after phagocytosis. Conversely, exogenous polyP prevents the killing of ingested bacteria that are normally killed after phagocytosis by human macrophages and the eukaryotic microbe Dictyostelium discoideum. This suggests the possibility that in these cells, a signal transduction pathway is used to sense polyP and prevent killing of ingested bacteria. In this report, we identify key components of the polyP signal transduction pathway in D. discoideum. In cells lacking these components, polyP is unable to inhibit killing of ingested bacteria. The pathway components have orthologs in human cells, and an exciting possibility is that pharmacologically blocking this pathway in human macrophages would cause them to kill ingested pathogens such as Mycobacterium tuberculosis.

Quantum chemical calculations of nicotine and caffeine molecule in gas phase and solvent using DFT methods.

In this study, the molecular structures of nicotine and caffeine molecule have been generated using the 6-311++G(d,p) basis set in the DFT/B3LYP method. The molecules were optimized on the same basis set and their minimum stable energy was calculated. The HOMO-LUMO energies were calculated to establish the kinetic stability and chemical reactivity of the chosen compounds. The variation of energy and its gap were closely studied for both nicotine and caffeine in the presence of solvent water as well. Similarly, vibrational spectroscopy was studied at the most prominent region in both gas phase and solvent water with their respective TED assignments. The shifting of frequency clearly indicates the impact of solvent water and isotopic substitution of carbon atoms.

Identification of novel proteins in the Dictyostelium discoideum chemorepulsion pathway using REMI.

Chemorepulsion, the biased migration of a cell away from a signal, is essential for many biological processes and the ability to manipulate chemorepulsion could lead to new therapeutics for a variety of diseases. However, little is known about eukaryotic cell chemorepulsion. Utilizing the model organism Dictyostelium discoideum, we previously identified an endogenous chemorepellent protein secreted by D. discoideum cells called AprA, and proteins involved in the AprA-induced chemorepulsion pathway including the G protein-coupled receptor GrlH, G beta and G protein alpha 8 protein subunits, protein kinase A, components of the mammalian target of rapamycin complex 2 (mTORC2), phospholipase A, PTEN and a PTEN-like phosphatase (CnrN), a retinoblastoma orthologue (RblA), extracellular signal-regulated kinase 1 (Erk1), p-21 activated protein kinase D (PakD), and the Ras proteins RasC and RasG. In this report, we used a genetic screen to identify 17 additional proteins involved in the AprA-induced chemorepulsion pathway .

Using Dictyostelium to Develop Therapeutics for Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) involves damage to lungs causing an influx of neutrophils from the blood into the lung airspaces, and the neutrophils causing further damage, which attracts more neutrophils in a vicious cycle. There are ∼190,000 cases of ARDS per year in the US, and because of the lack of therapeutics, the mortality rate is ∼40%. Repelling neutrophils out of the lung airspaces, or simply preventing neutrophil entry, is a potential therapeutic. In this minireview, we discuss how our lab noticed that a protein called AprA secreted by growing Dictyostelium cells functions as a repellent for Dictyostelium cells, causing cells to move away from a source of AprA. We then found that AprA has structural similarity to a human secreted protein called dipeptidyl peptidase IV (DPPIV), and that DPPIV is a repellent for human neutrophils. In animal models of ARDS, inhalation of DPPIV or DPPIV mimetics blocks neutrophil influx into the lungs. To move DPPIV or DPPIV mimetics into the clinic, we need to know how this repulsion works to understand possible drug interactions and side effects. Combining biochemistry and genetics in Dictyostelium to elucidate the AprA signal transduction pathway, followed by drug studies in human neutrophils to determine similarities and differences between neutrophil and Dictyostelium chemorepulsion, will hopefully lead to the safe use of DPPIV or DPPIV mimetics in the clinic.

Domain Organization of the UBX Domain Containing Protein 9 and Analysis of Its Interactions With the Homohexameric AAA + ATPase p97 (Valosin-Containing Protein).

The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum. Pull-down assays revealed that the UBX domain (amino acids 384-466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a ß0-α- 1-α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261-573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261-573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1' intermolecular interface. We thus propose that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.

An Autocrine Negative Feedback Loop Inhibits Dictyostelium discoideum Proliferation through Pathways Including IP3/Ca2.

Little is known about how eukaryotic cells can sense their number or spatial density and stop proliferating when the local density reaches a set value. We previously found that Dictyostelium discoideum accumulates extracellular polyphosphate to inhibit its proliferation, and this requires the G protein-coupled receptor GrlD and the small GTPase RasC. Here, we show that cells lacking the G protein component Gß, the Ras guanine nucleotide exchange factor GefA, phosphatase and tensin homolog (PTEN), phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP3) receptor-like protein A (IplA), polyphosphate kinase 1 (Ppk1), or the TOR complex 2 component PiaA have significantly reduced sensitivity to polyphosphate-induced proliferation inhibition. Polyphosphate upregulates IP3, and this requires GrlD, GefA, PTEN, PLC, and PiaA. Polyphosphate also upregulates cytosolic Ca2+, and this requires GrlD, Gß, GefA, RasC, PLC, IplA, Ppk1, and PiaA. Together, these data suggest that polyphosphate uses signal transduction pathways including IP3/Ca2+ to inhibit the proliferation of D. discoideum. IMPORTANCE Many mammalian tissues such as the liver have the remarkable ability to regulate their size and have their cells stop proliferating when the tissue reaches the correct size. One possible mechanism involves the cells secreting a signal that they all sense, and a high level of the signal tells the cells that there are enough of them and to stop proliferating. Although regulating such mechanisms could be useful to regulate tissue size to control cancer or birth defects, little is known about such systems. Here, we use a microbial system to study such a mechanism, and we find that key elements of the mechanism have similarities to human proteins. This then suggests the possibility that we may eventually be able to regulate the proliferation of selected cell types in humans and animals.

Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum.

Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy. Dictyostelium discoideumatg5 single, atg5/12 double, and atg5/12/16 triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on Klebsiella aerogenes was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of Escherichia coli was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of E. coli, was seen for ATG5¯ and ATG5¯/12¯/16¯ cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.

An endogenous chemorepellent directs cell movement by inhibiting pseudopods at one side of cells.

Eukaryotic chemoattraction signal transduction pathways, such as those used by Dictyostelium discoideum to move toward cAMP, use a G protein-coupled receptor to activate multiple conserved pathways such as PI3 kinase/Akt/PKB to induce actin polymerization and pseudopod formation at the front of a cell, and PTEN to localize myosin II to the rear of a cell. Relatively little is known about chemorepulsion. We previously found that AprA is a chemorepellent protein secreted by Dictyostelium cells. Here we used 29 cell lines with disruptions of cAMP and/or AprA signal transduction pathway components, and delineated the AprA chemorepulsion pathway. We find that AprA uses a subset of chemoattraction signal transduction pathways including Ras, protein kinase A, target of rapamycin (TOR), phospholipase A, and ERK1, but does not require the PI3 kinase/Akt/PKB and guanylyl cyclase pathways to induce chemorepulsion. Possibly as a result of not using the PI3 kinase/Akt/PKB pathway and guanylyl cyclases, AprA does not induce actin polymerization or increase the pseudopod formation rate, but rather appears to inhibit pseudopod formation at the side of cells closest to the source of AprA.

Extracellular signaling in Dictyostelium.

In the last few decades, we have learned a considerable amount about how eukaryotic cells communicate with each other, and what it is the cells are telling each other. The simplicity of Dictyostelium discoideum, and the wide variety of available tools to study this organism, makes it the equivalent of a hydrogen atom for cell and developmental biology. Studies using Dictyostelium have pioneered a good deal of our understanding of eukaryotic cell communication. In this review, we will present a brief overview of how Dictyostelium cells use extracellular signals to attract each other, repel each other, sense their local cell density, sense whether the nearby cells are starving or stressed, count themselves to organize the formation of structures containing a regulated number of cells, sense the volume they are in, and organize their multicellular development. Although we are probably just beginning to learn what the cells are telling each other, the elucidation of Dictyostelium extracellular signals has already led to the development of possible therapeutics for human diseases.

Functional Characterization of Ubiquitin-Like Core Autophagy Protein ATG12 in Dictyostelium discoideum.

Autophagy is a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagy protein ATG12 plays, together with ATG5 and ATG16, an essential role in the expansion of the autophagosomal membrane. In this study we analyzed gene replacement mutants of atg12 in Dictyostelium discoideum AX2 wild-type and ATG16‾ cells. RNAseq analysis revealed a strong enrichment of, firstly, autophagy genes among the up-regulated genes and, secondly, genes implicated in cell motility and phagocytosis among the down-regulated genes in the generated ATG12‾, ATG16‾ and ATG12‾/16‾ cells. The mutant strains showed similar defects in fruiting body formation, autolysosome maturation, and cellular viability, implying that ATG12 and ATG16 act as a functional unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12 and ATG16 resulted in slightly more severe defects in axenic growth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional function in these processes. Phagocytosis of yeast, spore viability, and maximal cell density were much more affected in ATG12‾/16‾ cells, indicating that both proteins also have cellular functions independent of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.