Use of pHlurorin-mKate2-human LC3 to Monitor Autophagic Responses.

In this chapter, we introduce the usage of pHluorin-mKate2-human LC3 for monitoring autophagy. Using EGFP and RFP, tandem fluorescent protein-tagged LC3 has been generated for monitoring autophagic structures. A critical point for this purpose is the sensitivity of the green fluorescent protein to acidic pH. A super-ecliptic pHluorin is most sensitive to acidic pH among EGFP, mWasabi, and pHluorin, indicating pHluorin is most suitable for monitoring autophagic structures. During autophagy, green-positive and red-positive fluorescent puncta of pHluorin-mKate2-human LC3 indicate signals of preautophagosomes and autophagosomes. After fusion of autophagosomes with lysosomes to form autolysosomes, green fluorescence of this intraautophagosomal protein is abolished according to acidification of autolysosomes. Therefore, these green-negative and red-positive fluorescent puncta reflect autolysosomes, in which intraluminal proteins are finally degraded by lysosomal proteases. To monitor autophagic flux, the accumulation of its green-negative and red-positive fluorescent puncta is monitored by inhibiting major lysosomal proteases, cathepsins. In addition, a mutant pHluorin-mKate2-human LC3△G is also introduced as a negative control probe.

ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation.

Expanded polyglutamine 72 repeat (polyQ72) aggregates induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and vesicular formation (autophagy). We examined this relationship and the molecular mechanism of autophagy formation. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent microtubule-associated protein 1 (MAP1) light chain 3 (LC3) conversion from LC3-I to -II, which plays a key role in autophagy. The eucaryotic translation initiation factor 2 alpha (eIF2alpha) A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, autophagy formation is a cellular defense mechanism against polyQ72-induced ER-stress-mediated cell death by degrading polyQ72 aggregates, with PERK/eIF2alpha phosphorylation being involved in polyQ72-induced LC3 conversion.

The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation.

Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p conjugation system and the Apg8p membrane targeting in autophagy and cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae. Similar to the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like enzyme, followed by conjugation with phosphatidylethanolamine. Evidence is presented here that Apg7p forms a homodimer with two active-site cysteine residues via the C-terminal region. The dimerization of Apg7p is independent of the other Apg proteins and facilitated by overexpressed Apg12p. The C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is neither an ATP binding domain nor an active-site cysteine essential for its E1 activity. The deletion of its carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p. These results suggest that Apg7p forms a homodimer via the C-terminal region and that the C-terminal region is essential for both the activity of the E1 enzyme for Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p.

The human homolog of Saccharomyces cerevisiae Apg7p is a Protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3.

Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system. In the yeast, Saccharomyces cerevisiae, an autophagosome is formed in the cytosol. The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole. The autophagic body is subsequently degraded by vacuolar hydrolases. Taking advantage of yeast genetics, apg (autophagy-defective) mutants were isolated that are defective in terms of formation of autophagic bodies under nutrient starvation conditions. One of the APG gene products, Apg12p, is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation, and this conjugation is essential for autophagy. Apg7p is a novel E1 enzyme essential for the Apg12p-conjugation system. In mammalian cells, the human Apg12p homolog (hApg12p) also conjugates with the human Apg5p homolog. In this study, the unique characteristics of hApg7p are shown. A two-hybrid experiment indicated that hApg12p interacts with hApg7p. Site-directed mutagenesis revealed that Cys(572) of hApg7p is an authentic active site cysteine residue essential for the formation of the hApg7p.hApg12p intermediate. Overexpression of hApg7p enhances the formation of the hApg5p.hApg12p conjugate, indicating that hApg7p is an E1-like enzyme essential for the hApg12p conjugation system. Cross-linking experiments and glycerol-gradient centrifugation analysis showed that the mammalian Apg7p homolog forms a homodimer as in yeast Apg7p. Each of three human Apg8p counterparts, i.e. the Golgi-associated ATPase enhancer of 16 kDa, GABA(A) receptor-associated protein, and microtubule-associated protein light chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant hApg7p(C572S) to form a stable intermediate via an ester bond. These results indicate that hApg7p is an authentic protein-activating enzyme for hApg12p and the three Apg8p homologs.

A ubiquitin-like system mediates protein lipidation.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.

A lysosomal proteinase, the late infantile neuronal ceroid lipofuscinosis gene (CLN2) product, is essential for degradation of a hydrophobic protein, the subunit c of ATP synthase.

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.

Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy.

In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.

Autolysosomal membrane-associated betaine homocysteine methyltransferase. Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy.

We compared the membrane proteins of autolysosomes isolated from leupeptin-administered rat liver with those of lysosomes. In addition to many polypeptides common to the two membranes, the autolysosomal membranes were found to be more enriched in endoplasmic reticulum lumenal proteins (protein-disulfide isomerase, calreticulin, ER60, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58-kDa protein) than lysosomal membranes. The autolysosomal membrane proteins include three polypeptides (44, 35, and 32 kDa) whose amino-terminal sequences have not yet been reported. Combining immunoblotting and reverse transcriptase-polymerase chain reaction analyses, we identified the 44-kDa peptide as the intact subunit of betaine homocysteine methyltransferase and the 35- and 32-kDa peptides as two proteolytic fragments. Pronase digestion of autolysosomes revealed that the 44-kDa and 32-kDa peptides are present in the lumen, whereas the 35-kDa peptide is not. In primary hepatocyte cultures, the starvation-induced accumulation of the 32-kDa peptide occurs in the presence of E64d, showing that the 32-kDa peptide is formed from the sequestered 44-kDa peptide during autophagy. The accumulation is induced by rapamycin but completely inhibited by wortmannin, 3-methyladenine, and bafilomycin. Thus, detection of the 32-kDa peptide by immunoblotting can be used as a streamlined assay for monitoring autophagy.

Yeast Cls2p/Csg2p localized on the endoplasmic reticulum membrane regulates a non-exchangeable intracellular Ca2+ pool cooperatively with calcineurin.

Saccharromyces cerevisiae CLS2 gene product (Cls2p) that is localized on the endoplasmic reticulum is important for the regulation of intracellular Ca2+ in a compartment distinct from the vacuole. Using a vma3 mutation that impairs the Ca2+ sequestering activity into the vacuole, we have shown that the cls2 mutation results in 3.4-fold increase in the Ca2+ pool that is not exchangeable with extracellular Ca2+. Accumulation of Ca2+ within the cls2 cells is synergistically elevated by the addition of immunosuppressant, FK506. Moreover, in the vma3 background, toxicity caused by the cls2 mutation is greatly enhanced by FK506. Given that FK506 inhibits the calcineurin activity, Cls2p likely functions in releasing Ca2+ flux from the endoplasmic reticulum, somehow cooperating with calcineurin.

Cooperation of calcineurin and vacuolar H(+)-ATPase in intracellular Ca2+ homeostasis of yeast cells.

Saccharomyces cerevisiae VMA genes, encoding essential components for the expression of vacuolar membrane H(+)-ATPase activity, are involved in intracellular ionic homeostasis and vacuolar biogenesis. We report here that the immunosuppressants FK506 and cyclosporin A cause general growth inhibition of the vma3 mutant. Upon addition of the drugs, the mutant grew neither in the presence of more than 5 mM Ca2+ nor above pH 6.0. The action of the immunosuppressants is dependent on their binding proteins and ascribable to inhibition of calcineurin activity; a mutation of a calcineurin subunit (cnb1) shows synthetic lethal interaction with the vma mutation. The addition of FK506 decreases the cytosolic free concentration of Ca2+ in the vma3 mutant cells. Consequently, FK506 induces an 8.9-fold elevation of a nonexchangeable Ca2+ pool. These results suggest that calcineurin controls calcium homeostasis by repression of Ca2+ flux into a cellular compartment(s) and that the vacuolar H(+)-ATPase is essential for cell growth cooperating with calcineurin to regulate the cytosolic free concentration of Ca2+.

Functional dissection of a rice high-pI alpha-amylase gene promoter.

Deletion analysis has previously shown that a 260 bp fragment, located between positions -230 and +29 of the 5' end of a rice high-pI alpha--amylase gene, OSamy-c, is required for gibberellic acid (GA3)-dependent transcriptional activation. We have since established a quantitative transient assay based on expression of a luciferase reporter gene in rice aleurone cells and continued to characterize the OSamy-c promoter for GA3-dependent regulatory sequences. Using this method, we have shown that the DNA sequence between -158 and -46 (sequence I) is sufficient to confer GA3-responsive activation on OSamy-c. We have also shown that this sequence is capable of directing GA3-dependent expression from a heterologous minimal promoter. Our results also showed that sequence I confers GA3 regulatory control in an orientation-dependent manner and interacts with two further upstream DNA sequences, II and III, in a combination which mildly enhances the level of the GA3 response exhibited by sequence I. Thus, we propose that sequence I confers the fundamental GA3-responsive character on OSamy-c, and that regulatory proteins that bind sequences II and III interact with each other and with regulatory proteins that bind sequence I, effectively to modulate the GA3 response.

High-level expression of the truncated alpha chain of human high-affinity receptor for IgE as a soluble form by baculovirus-infected insect cells. Biochemical characterization of the recombinant product.

The binding subunit of human high-affinity receptor for IgE (Fc epsilon RI alpha) was efficiently expressed as a truncated form in insect cells. The soluble (s)Fc epsilon RI alpha purified from culture medium by affinity chromatography with an anti-(alpha chain) mAb was nearly homogeneous and had an IgE-binding activity. The amino acid composition and the revealed N-terminal amino acid sequence of sFc epsilon RI alpha suggested that it was properly processed in insect cells. The apparent molecular mass (35 kDa) of purified sFc epsilon RI alpha was smaller than that of sFc epsilon RI alpha produced by CHO transfectants. The reduction of the apparent molecular mass after N-glycanase treatment showed the recombinant product was N-glycosylated. Peptide mapping of native and deglycosylated sFc epsilon RI alpha indicated that three Asn residues (Asn21, Asn42 and Asn166) should be almost fully glycosylated, and that two Asn residues (Asn74 and Asn135) were partially glycosylated.

Calcium-sensitive cls mutants of Saccharomyces cerevisiae showing a Pet- phenotype are ascribable to defects of vacuolar membrane H(+)-ATPase activity.

Ca(2+)-sensitive mutants of the yeast Saccharomyces cerevisiae showing a Pet- phenotype (cls7-cls11) have lesions in a system for maintaining intracellular Ca2+ homeostasis (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1986) J. Gen. Microbiol. 132, 979-988). Genetic and biochemical studies have demonstrated that these Pet- cls mutants are related to defects in vacuolar membrane H(+)-ATPase. CLS7 and CLS8 were found to be identical with the structural genes encoding subunit c (VMA3) and subunit a (VMA1), respectively, of the enzyme. In addition, these five mutants all had vma defects; no vacuolar membrane ATPase activity was detected in the cls cells, and the cls mutants showed a loss of ability to acidify the vacuole in vivo. Measurements of the cytosolic free Ca2+ concentration [( Ca2+]i) in individual cells showed that the average [Ca2+]i in wild-type cells was 150 +/- 80 nM, whereas that in five Pet- cls cells was 900 +/- 100 nM. These data are consistent with the observation that vacuolar membrane vesicles prepared from the Pet- cls cells have lost ATP-dependent Ca2+ uptake activities. The cls defects of vacuolar membrane H(+)-ATPase resulted in pleiotropic effects on several cellular activities, including Ca2+ homeostasis, glycerol metabolism, and phospholipid metabolism. The mutants showed an inositol-dependent phenotype, possibly due to alteration in regulation of phospholipid biosynthesis; the phosphatidylserine decarboxylase activities of the mutants were 15-50% of that of the wild-type cells and were not repressed by the addition of inositol. In contrast to the majority of previously isolated pet mutants (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225), the Pet- cls mutants showed no detectable mitochondrial defects. Taking all these findings into account, we suggest that at least six genes, VMA1 (CLS8, subunit a), VMA2 (subunit b), VMA3 (CLS7, subunit c), VMA11 (CLS9), VMA12 (CLS10), and VMA13 (CLS11), are required for expression of the vacuolar membrane H(+)-ATPase activity.

A multinuclear magnetic resonance study of a cls11 mutant showing the Pet- phenotype of Saccharomyces cerevisiae.

Energetic and intermediary metabolism was studied in a Pet- mutant of Saccharomyces cerevisiae with a calcium-sensitive phenotype that shows an inability to grow when cultured in a medium containing non-fermentable substrates. The perchloric acid extracts were prepared from suspensions of cls11 mutant and wild-type cells incubated with [1,3-13C]glycerol or [2-13]acetate, and analyzed by 31P, 13C and 1H NMR. 31P- and 1H-NMR spectra showed significant differences between cls11 and wild-type cells at the level of amino acids, the storage carbohydrate trehalose (higher in mutant cells), and sugar phosphates (higher in wild-type cells). 13C-NMR spectra revealed major differences in the steady-state labelling of glutamate carbons. For incubations with [1,3-13C]glycerol, we estimated from the relative 13C enrichment of glutamate carbons that acetyl-CoA C2 is 43% C13 labelled in wild-type and 10% 13C labelled in mutant cells, respectively. For incubations with [2-13C]acetate, we calculated that the ratio of the relative flux through the glyoxylate shunt versus oxidative reactions is 58% in wild-type cells and 44% in the cls11 mutant cells. Again, a dilution of the relative enrichment of C2 of acetyl-CoA was observed in the mutant cells (89%) compared to the wild-type cells (97%). These results are discussed in terms of pleiotropic defects in non-fermentable carbon metabolism in mutant cells.

Collapse of cortex expansion during germination of Bacillus megaterium spores.

When spores of Bacillus megaterium ATCC 12872 were incubated with CdCl2, they germinated without decomposition of the cortex. Moreover, the volume ratio of cortex to protoplast-plus-cortex, C/(P+C), of the CdCl2-germinated spores was reduced. Incubation of isolated cortex with the divalent compounds Cd2+, Ca2+, and Mg2+ reduced the gel volume to about 1/5 but incubation with a nonionic compounds, glucose, did not. The spores with reduced C/(P+C) were observed in the early period of glucose-induced germination. The time required for a 50% change in cortex morphology to occur was 2.5 min, which corresponds well with the time for 50% loss of heat resistance. This time was shorter than that necessary for release of peptidoglycan fragments and hydrolysis of cortex glycan chains. These data indicate that cortex hydrolysis is not related to the initiation of germination. 50% of the dipicolinic acid, calcium and magnesium were released at 3.4, 4.0, and 2.4 min, respectively. These results suggest that collapse of cortex expansion by the interaction of cortex with dipicolinic acid and cations released from the core, or exogenous ionic germinants is an important step in the initiation of germination.