Unlocking Nature's Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter.

BACKGROUND: Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (PPEPCK) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins. RESULTS: PPEPCK-RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from PPEPCK. The traditional, methanol-inducible alcohol oxidase 1 promoter (PAOX1) was used as the benchmark. Initial studies carried out with MSG as the inducer resulted in low recombinant protein yield. A new strategy employing MSG/ethanol mixed feeding improved biomass generation as well as recombinant protein yield. Cell density of 100-120 A600 units/ml was achieved after 72 h of induction in shake flask cultivations, resulting in recombinant protein yield from PPEPCK that is comparable or even higher than that from PAOX1. CONCLUSIONS: We have designed an induction medium for recombinant protein production from K. phaffii PPEPCK in shake flask cultivations. It consists of 1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate (pH 6.0), 0.4 mg/L biotin, 2.0% MSG, and 2% ethanol. Substitution of ammonium sulphate with 0.5% urea is optional. Carbon source was replenished every 24 h during 72 h induction period. Under these conditions, GFP and RBD yields from PPEPCK equaled and even surpassed those from PAOX1. Compared to the traditional methanol-inducible expression system, the inducers of glutamate-inducible expression system are non-toxic and their metabolism does not generate toxic metabolites such as formaldehyde and hydrogen peroxide. This study sets the stage for MSG-inducible, industrial scale recombinant protein production from K. phaffii PPEPCK in bioreactors.

Characterization of the transactivation and nuclear localization functions of Pichia pastoris zinc finger transcription factor Mxr1p.

The zinc finger transcription factor Mxr1p regulates the transcription of genes involved in methanol, acetate, and amino acid metabolism of the industrial yeast Pichia pastoris (a.k.a. Komagataella phaffii) by binding to Mxr1p response elements in their promoters. Here, we demonstrate that Mxr1p is a key regulator of ethanol metabolism as well. Using transcriptomic analysis, we identified target genes of Mxr1p that mediate ethanol metabolism, including ALD6-1 encoding an aldehyde dehydrogenase. ALD6-1 is essential for ethanol metabolism, and the ALD6-1 promoter harbors three Mxr1p response elements to which Mxr1p binds in vitro and activates transcription in vivo. We show that a nine-amino acid transactivation domain located between amino acids 365 and 373 of Mxr1p is essential for the transactivation of ALD6-1 to facilitate ethanol metabolism. Mxr1N250, containing the N-terminal 250 amino acids of Mxr1p, localized to the nucleus of cells metabolizing ethanol dependent on basic amino acid residues present between amino acids 75 and 85. While the N-terminal 400 amino acids of Mxr1p are sufficient for the activation of target genes essential for ethanol metabolism, the region between amino acids 401 and 1155 was also required for the regulation of genes essential for methanol metabolism. Finally, we identified several novel genes whose expression is differentially regulated by Mxr1p during methanol metabolism by DNA microarray. This study demonstrates that Mxr1p is a key regulator of ethanol metabolism and provides new insights into the mechanism by which Mxr1p functions as a global regulator of multiple metabolic pathways of P. pastoris.

Posttranscriptional regulation of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase in Komagataella phaffii.

The yeast Komagataella phaffii (a.k.a. Pichia pastoris) harbours a unique glutamate utilization pathway in which the cytosolic enzymes glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the sequential conversion of glutamate to α-ketoglutarate, oxaloacetate and phosphoenolpyruvate respectively. GDH2 and PEPCK are essential for glutamate catabolism. Their synthesis is induced by autophagy during carbon starvation and are essential for cell survival. Here, we demonstrate that GDH2 and PEPCK reciprocally regulate each other's protein levels during glutamate catabolism such that GDH2 is downregulated in Δpepck and PEPCK is downregulated in Δgdh2. We further demonstrate that sequential conversion of glutamate to α-ketoglutarate and oxaloacetate by GDH2 and AAT2, respectively, is essential for PEPCK synthesis in cells metabolizing glutamate. Our studies indicate that translation of GDH2 mRNA is induced by glutamate while oxaloacetate derived from glutamate is likely to be the inducer of PEPCK mRNA translation during glutamate catabolism. Thus, GDH2- and PEPCK-catalyzed reactions are essential for ATP generation and gluconeogenesis respectively during carbon starvation and glutamate catabolism in K. phaffii. We conclude that K. phaffii harbours a unique translational regulatory circuit in which substrates of GDH2 and PEPCK act as inducers of their synthesis, a phenomenon not reported in any yeast species.

Carbon starvation-induced synthesis of GDH2 and PEPCK is essential for the survival of Pichia pastoris.

The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs. GDH2 and PEPCK synthesis is abrogated in Δatg1 indicating a key role for autophagy or an autophagy-related process. Finally, carbon-starved Δgdh2 and Δpepck exhibit poor survival. This study demonstrates a key role for amino acid catabolism during carbon starvation, a phenomenon hitherto unreported in other yeast species.

The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris.

Rtg1p and Rtg3p are two basic helix-loop-helix, retrograde transcription factors in the budding yeast Saccharomyces cerevisiae Both factors heterodimerize to activate the transcription of nuclear genes in response to mitochondrial dysfunction and glutamate auxotrophy, but are not well characterized in other yeasts. Here, we demonstrate that the Rtg1p/Rtg3p-mediated retrograde signaling pathway is absent in the methylotrophic yeast Pichia pastoris We observed that P. pastoris Rtg1p (PpRtg1p) heterodimerizes with S. cerevisiae Rtg3p and functions as a nuclear, retrograde transcription factor in S. cerevisiae, but not in P. pastoris. We noted that P. pastoris Rtg3p lacks a functional leucine zipper and interacts with neither S. cerevisiae Rtg1p (ScRtg1p) nor PpRtg1p. In the absence of an interaction with Rtg3p, PpRtg1p has apparently acquired a novel function as a cytosolic regulator of multiple P. pastoris metabolic pathways, including biosynthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase required for the utilization of glutamate as the sole carbon source. PpRtg1p also had an essential role in methanol metabolism and regulated alcohol oxidase synthesis and was required for the metabolism of ethanol, acetate, and oleic acid, but not of glucose and glycerol. Although PpRtg1p could functionally complement ScRtg1p, ScRtg1p could not complement PpRtg1p, indicating that ScRtg1p is not a functional PpRtg1p homolog. Thus, PpRtg1p functions as a nuclear, retrograde transcription factor in S. cerevisiae and as a cytosolic, post-transcriptional regulator in P. pastoris We conclude that PpRtg1p is a key component of a signaling pathway that regulates multiple metabolic processes in P. pastoris.

Methionine synthase is localized to the nucleus in Pichia pastoris and Candida albicans and to the cytoplasm in Saccharomyces cerevisiae.

Methionine synthase (MS) catalyzes methylation of homocysteine, the last step in the biosynthesis of methionine, which is essential for the regeneration of tetrahydrofolate and biosynthesis of S-adenosylmethionine. Here, we report that MS is localized to the nucleus of Pichia pastoris and Candida albicans but is cytoplasmic in Saccharomyces cerevisiae The P. pastoris strain carrying a deletion of the MET6 gene encoding MS (Ppmet6) exhibits methionine as well as adenine auxotrophy indicating that MS is required for methionine as well as adenine biosynthesis. Nuclear localization of P. pastoris MS (PpMS) was abrogated by the deletion of 107 C-terminal amino acids or the R742A mutation. In silico analysis of the PpMS structure indicated that PpMS may exist in a dimer-like configuration in which Arg-742 of a monomer forms a salt bridge with Asp-113 of another monomer. Biochemical studies indicate that R742A as well as D113R mutations abrogate nuclear localization of PpMS and its ability to reverse methionine auxotrophy of Ppmet6 Thus, association of two PpMS monomers through the interaction of Arg-742 and Asp-113 is essential for catalytic activity and nuclear localization. When PpMS is targeted to the cytoplasm employing a heterologous nuclear export signal, it is expressed at very low levels and is unable to reverse methionine and adenine auxotrophy of Ppmet6 Thus, nuclear localization is essential for the stability and function of MS in P. pastoris. We conclude that nuclear localization of MS is a unique feature of respiratory yeasts such as P. pastoris and C. albicans, and it may have novel moonlighting functions in the nucleus.

Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris.

Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB + Glu), or YNB + aspartate (YNB + Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB + Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB + Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB + Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1 We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris.

Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris.

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris.

Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein.

Mouse Apolipoprotein L9 (ApoL9) is an understudied cytoplasmic, interferon-inducible protein. The details of its intracellular localization and normal cellular functions are unclear. We report here that ApoL9 localizes to small puncta diffusely distributed in the cytoplasm, as well as to larger granules of varying size and number that are similar to aggresome-like induced structures (ALIS) and contain the autophagy receptor Sqstm1/p62, the autophagosome marker Lc3, and ubiquitin. Transfection of B16F10 mouse melanoma cells stably expressing ApoL9 (B16F10L9) with certain liposome-based transfection reagents causes dramatic disturbances in its subcellular distribution. We reasoned that these disturbances may be due to the interaction of ApoL9 with dioleoylphosphatidylethanolamine (DOPE), the helper lipid component of several transfection reagents. Recombinant ApoL9 produced in E. coli, as well as ApoL9 expressed in HEK293T cells, specifically bind phosphatidylethanolamine (PE) in vitro. ApoL9 is expressed at high levels in liver and brain, organs enriched in PE. Since PE is known to facilitate replication of positive strand RNA viruses, we examined the role of ApoL9 during replication of Japanese encephalitis virus (JEV), a positive strand virus of the family Flaviviridae. JEV titres in B16F10L9 cells are higher than those in B16F10 cells. We propose that ApoL9 is a PE-binding protein that may have important roles in several cellular processes that involve this phospholipid.

Trm1p, a Zn(II)2Cys6-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris.

The zinc finger transcription factors Mxr1p and Rop are key regulators of methanol metabolism in the methylotrophic yeast, Pichia pastoris, while Trm1p and Trm2p regulate methanol metabolism in Candida boidinii. Here, we demonstrate that Trm1p is essential for the expression of genes of methanol utilization (mut) pathway in P. pastoris as well. Expression of AOXI and other genes of mut pathway is severely compromised in P. pastoris ΔTrm1 strain resulting in impaired growth on media containing methanol as the sole source of carbon. Trm1p localizes to the nucleus of cells cultured on glucose or methanol. The zinc finger domain of Mxr1p but not Trm1p binds to AOXI promoter sequences in vitro, indicating that these two positive regulators act by different mechanisms. We conclude that both Trm1p and Mxr1p are essential for the expression of genes of mut pathway in P. pastoris and the mechanism of transcriptional regulation of mut pathway may be similar in P. pastoris and C. boidinii.

The zinc finger proteins Mxr1p and repressor of phosphoenolpyruvate carboxykinase (ROP) have the same DNA binding specificity but regulate methanol metabolism antagonistically in Pichia pastoris.

The methanol-inducible alcohol oxidase I (AOXI) promoter of the methylotrophic yeast, Pichia pastoris, is used widely for the production of recombinant proteins. AOXI transcription is regulated by the zinc finger protein Mxr1p (methanol expression regulator 1). ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) is a methanol- and biotin starvation-inducible zinc finger protein that acts as a negative regulator of PEPCK in P. pastoris cultured in biotin-deficient, glucose-ammonium medium. The function of ROP during methanol metabolism is not known. In this study, we demonstrate that ROP represses methanol-inducible expression of AOXI when P. pastoris is cultured in a nutrient-rich medium containing yeast extract, peptone, and methanol (YPM). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion whereas overexpression of ROP results in repression of AOXI and growth retardation of P. pastoris cultured in YPM medium. Surprisingly, deletion or overexpression of ROP has no effect on AOXI gene expression and growth of P. pastoris cultured in a minimal medium containing yeast nitrogen base and methanol (YNBM). Subcellular localization studies indicate that ROP translocates from cytosol to nucleus of cells cultured in YPM but not YNBM. In vitro DNA binding studies indicate that AOXI promoter sequences containing 5' CYCCNY 3' motifs serve as binding sites for Mxr1p as well as ROP. Thus, Mxr1p and ROP exhibit the same DNA binding specificity but regulate methanol metabolism antagonistically in P. pastoris. This is the first report on the identification of a transcriptional repressor of methanol metabolism in any yeast species.

An alternative model for heme biosynthesis in the malarial parasite.

The malarial parasite imports an infected host's red blood cell enzymes for heme biosynthesis during the intraerythrocytic stage. This is despite all the genes of the heme-biosynthetic pathway having been identified on the parasite genome. On the basis of predictions of parasite genome-coded enzyme localization, functionality of some of these enzymes and shuttling of intermediates between different parasite compartments, a hybrid model for parasite heme biosynthesis has been proposed. However, this model does not take into account the possible role of imported host enzymes in parasite heme biosynthesis. We propose an alternative model with an extrinsic heme-biosynthetic pathway in the parasite cytosol that uses imported host enzymes, and an intrinsic pathway confined to the organellar fractions that uses the parasite-genome-encoded enzymes.

Histidine is essential for growth of Komagataella phaffii cultured in YPA medium.

Komagataella phaffii (a.k.a. Pichia pastoris) requires histidine for optimal growth when cultured in a medium containing yeast extract, peptone (YP), and acetate (YPA). We demonstrate that HIS4-deficient, K. phaffii strain GS115 exhibits a growth defect on YP-media containing acetate, but not on other carbon sources. K. phaffii X33, a prototroph, grows better than K. phaffii GS115 (his4), a histidine auxotroph in YPA. Normal growth of GS115 is restored either by the expression of HIS4 or by culturing in YPA containing ≥0.6 mM histidine. In the presence of histidine, expression of several genes is altered, including those encoding key subunits of mitochondrial ATP synthase, transporters of amino acids and nutrients, as well as biosynthetic enzymes. Thus, histidine should be included as an essential component for optimal growth of K. phaffii histidine auxotrophs cultured in YPA.

Coordinate regulation of methanol utilization pathway genes of Komagataella phaffii by transcription factors and chromatin modifiers.

The methylotrophic yeast Komagataella phaffii (a.k.a. Pichia pastoris) harbors a methanol utilization (MUT) pathway, enabling it to utilize methanol as the sole source of carbon. The nexus between transcription factors such as Mxr1p and Trm1p and chromatin-modifying enzymes in the regulation of genes of MUT pathway has not been well studied in K. phaffii. Using transcriptomics, we demonstrate that Gcn5, a histone acetyltransferase, and Gal83, one of the beta subunits of nuclear-localized SNF1 (sucrose non-fermenting 1) kinase complex are essential for the transcriptional regulation by the zinc finger transcription factors Mxr1p and Trm1p. We conclude that interactions among Gcn5, Snf1, Mxr1p, and Trm1p play a critical role in the transcriptional regulation of genes of MUT pathway of K. phaffii.

Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris.

We have identified a methanol- and biotin-starvation-inducible zinc finger protein named ROP [repressor of phosphoenolpyruvate carboxykinase (PEPCK)] in the methylotrophic yeast Pichia pastoris. When P. pastoris strain GS115 (wild-type, WT) is cultured in biotin-deficient, glucose-ammonium (Bio(-)) medium, growth is suppressed due to the inhibition of anaplerotic synthesis of oxaloacetate, catalysed by the biotin-dependent enzyme pyruvate carboxylase (PC). Deletion of ROP results in a strain (ΔROP) that can grow under biotin-deficient conditions due to derepression of a biotin- and PC-independent pathway of anaplerotic synthesis of oxaloacetate. Northern analysis as well as microarray expression profiling of RNA isolated from WT and ΔROP strains cultured in Bio(-) medium indicate that expression of the phosphoenolpyruvate carboxykinase gene (PEPCK) is induced in ΔROP during biotin- or PC-deficiency even under glucose-abundant conditions. There is an excellent correlation between PEPCK expression and growth of ΔROP in Bio(-) medium, suggesting that ROP-mediated regulation of PEPCK may have a crucial role in the biotin- and PC-independent growth of the ΔROP strain. To our knowledge, ROP is the first example of a zinc finger transcription factor involved in the catabolite repression of PEPCK in yeast cells cultured under biotin- or PC-deficient and glucose-abundant conditions.

Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris.

Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E. coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOX1 promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.

Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris.

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris. Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N-terminal 150 amino acids, including the zinc finger DNA-binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5'-CYCC-3' sequences, although all DNA sequences containing the 5'-CYCC-3' motif do not qualify as Mxr1p-binding sites. Key DNA-binding determinants are present outside 5'-CYCC-3' motif and Mxr1p preferably binds to DNA sequences containing 5'-CYCCNY-3' than those containing 5'-CYCCNR-3' sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p-binding sites in other methanol-inducible promoters of P. pastoris.

Apolipoprotein L9 interacts with LC3/GABARAP and is a microtubule-associated protein with a widespread subcellular distribution.

Mouse Apolipoprotein L9 is a 34-kDa phosphatidylethanolamine (PE)-binding protein. The gene is present only in mouse and rat genomes; hence it is restricted to two species. To understand why, it is essential to uncover details about its functions in cellular processes. Here we show that ApoL9 interacts with the proteins of the LC3 and GABARAP subfamilies, which are key players in macroautophagy. In vitro binding studies show a strong association with GABARAP, and in amino acid-starved cells it preferentially interacts with lipidated LC3B, likely by binding to its PE moiety through its lipid-binding domain. On treatment with autophagy inhibitors bafilomycin A1 and chloroquine, ApoL9 is found near swollen mitochondria and on lysosomes/LAMP1-positive compartments. However, ApoL9 itself does not seem to be degraded as a result of autophagy, suggesting that it is not an autophagy cargo receptor. Deletions in a putative transmembrane region between amino acids 110 and 145 abolish binding to PE. In addition, ApoL9 can redistribute to stress granules, can homo-oligomerize, and is a microtubule-associated protein. In short, its distribution in the cell is quite widespread, suggesting that it could have functions at the intersection of membrane binding and reorganization, autophagy, cellular stress and intracellular lipid transport.This article has an associated First Person interview with the first author of the paper.

Isolation of a single-stranded DNA-binding protein from the methylotrophic yeast, Pichia pastoris and its identification as zeta crystallin.

A single-stranded DNA (ssDNA)-binding protein (SSB) that binds to specific upstream sequences of alcohol oxidase (AOX1) promoter of the methylotrophic yeast Pichia pastoris has been isolated and identified as zeta crystallin (ZTA1). The cDNA encoding P.pastoris ZTA1 (PpZTA1) was cloned into an Escherichia coli expression vector, the recombinant PpZTA1 was expressed and purified from E.coli cell lysates. The DNA-binding properties of recombinant PpZTA1 are identical to those of the SSB present in P.pastoris cell lysates. PpZTA1 binds to ssDNA sequences >24 nt and its DNA-binding activity is abolished by NADPH. This is the first report on the characterization of DNA-binding properties of a yeast ZTA1.

Curcumin as an Adjunct Drug for Infectious Diseases.

Curcumin, by virtue of its ability to function as an immunomodulator, has the potential to serve as an adjunct drug to treat infectious diseases and provide long-term protection. The current need is to establish clinical trials with curcumin as an adjunct drug against specific infectious diseases.