AP-3 Mutants Research Articles

A genome-wide copper-sensitized screen identifies novel regulators of mitochondrial cytochrome c oxidase activity

Copper is essential for the activity and stability of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Loss-of-function mutations in genes required for copper transport to CcO result in fatal human disorders. Despite the fundamental importance of copper in mitochondrial and organismal physiology, systematic identification of genes that regulate mitochondrial copper homeostasis is lacking. To discover these genes, we performed a genome-wide screen using a library of DNA-barcoded yeast deletion mutants grown in copper-supplemented media. Our screen recovered a number of genes known to be involved in cellular copper homeostasis as well as genes previously not linked to mitochondrial copper biology. These newly identified genes include the subunits of the adaptor protein 3 complex (AP-3) and components of the cellular pH-sensing pathway Rim20 and Rim21, both of which are known to affect vacuolar function. We find that AP-3 and Rim mutants exhibit decreased vacuolar acidity, which in turn perturbs mitochondrial copper homeostasis and CcO function. CcO activity of these mutants could be rescued by either restoring vacuolar pH or supplementing growth media with additional copper. Consistent with these genetic data, pharmacological inhibition of the vacuolar proton pump leads to decreased mitochondrial copper content and a concomitant decrease in CcO abundance and activity. Taken together, our study uncovered novel genetic regulators of mitochondrial copper homeostasis and provided a mechanism by which vacuolar pH impacts mitochondrial respiration through copper homeostasis.

The ADAPTOR PROTEIN-3 Complex Mediates Pollen Tube Growth by Coordinating Vacuolar Targeting and Organization.

Pollen tube growth is an essential step for successful plant reproduction. Vacuolar trafficking and dynamic organization are important for pollen tube growth; however, the key proteins involved in these processes are not well understood. Here, we report that the ADAPTOR PROTEIN-3 (AP-3) complex and its tonoplast cargo PROTEIN S-ACYL TRANSFERASE10 (PAT10) are critical for pollen tube growth in Arabidopsis (Arabidopsis thaliana). AP-3 is a heterotetrameric protein complex consisting of four subunits, δ, β, µ, and σ. AP-3 regulates tonoplast targeting of several cargoes, such as PAT10. We show that functional loss of any of the four AP-3 subunits reduces plant fertility. In ap-3 mutants, pollen development was normal but pollen tube growth was compromised, leading to reduced male transmission. Functional loss of PAT10 caused a similar reduction in pollen tube growth, suggesting that the tonoplast association of PAT10 mediated by AP-3 is crucial for this process. Indeed, the Ca2+ gradient during pollen tube growth was reduced significantly due to AP-3 loss of function, consistent with the abnormal targeting of CALCINUERIN B-LIKE2 (CBL2) and CBL3, whose tonoplast association depends on PAT10. Furthermore, we show that the pollen tubes of ap-3 mutants have vacuoles with simplified tubules and bulbous structures, indicating that AP-3 affects vacuolar organization. Our results demonstrate a role for AP-3 in plant reproduction and provide insights into the role of vacuoles in polarized cell growth.

Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization.

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9-positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP-3, which mediates direct transport from the trans-Golgi to the vacuole. The four C-terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP-3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.

Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant

Arabidopsis thaliana APETALA3 (AP3) and Antirrhinum majus DEFICIENS (DEF) MADS box genes are required to specify petal and stamen identity. AP3 and DEF are members of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. In order to investigate the molecular mechanisms underlying organ development in early diverging clades of core eudicots, we isolated and identified an AP3 homolog, FaesAP3, from Fagopyrum esculentum (buckwheat, Polygonaceae), a multi-food-use pseudocereal with healing benefits. Protein sequence alignment and phylogenetic analyses revealed that FaesAP3 grouped into the euAP3 lineage. Expression analysis showed that FaesAP3 was transcribed only in developing stamens, and differed from AP3 and DEF, which expressed in developing petals and stamens. Moreover, ectopic expression of FaesAP3 rescued stamen development without complementation of petal development in an Arabidopsis ap3 mutant. Our results suggest that FaesAP3 is involved in the development of stamens in buckwheat. These results also suggest that FaesAP3 holds some potential for biotechnical engineering to create a male sterile line of F. esculentum.

Two ancestral APETALA3 homologs from the basal angiosperm Magnolia wufengensis (Magnoliaceae) can affect flower development of Arabidopsis

APETALA3 (AP3) homologs are involved in specifying petal and stamen identities in core eudicot model organisms. In order to investigate the functional conservation of AP3 homologs between core eudicots and basal angiosperm, we isolated and identified two AP3 homologs from Magnolia wufengensis, a woody basal angiosperm belonging to the family Magnoliaceae. Sequence and phylogenetic analyses revealed that both genes are clade members of the paleoAP3 lineage. Moreover, a highly conserved motif of paleoAP3 is found in the C-terminal regions of MAwuAP3_1/2 proteins, but the PI-derived motif, usually present in AP3/DEF-like lineage members, is missing. Semi-quantitative and real time PCR analyses showed that the expression of MAwuAP3_1/2 was restricted to tepals and stamens. However, the MAwuAP3_1 expression was maintained at a high level during the rapid increased in size of tepals and stamens, while MAwuAP3_2 mRNA was only detected at the early stage of tepal and stamen development. Furthermore, the expression of MAwuAP3_1/2 in transgenic Arabidopsis causes phenotypic changes which partly resemble those caused by ectopic expressions of the endogenous AP3 gene. Moreover, the 35S::MAwuAP3_1/2 transgenic Arabidopsis can be used partially to rescue the loss-of-function ap3 mutant (ap3-3) of Arabidopsis. These findings call for a more comprehensive understanding of the B-functional evolution from basal angiosperm to core eudicot clades.

The paleoAP3-type gene CpAP3, an ancestral B-class gene from the basal angiosperm Chimonanthus praecox, can affect stamen and petal development in higher eudicots

Wintersweet (Chimonanthus praecox), a basal angiosperm endemic to China, has high ornamental value for developing beautiful flowers with strong fragrance. The molecular mechanism regulating flower development in wintersweet remains largely elusive. In this project, we seek to determine the molecular features and expression patterns of the C. praecox paleoAP3-type gene CpAP3 and examine its potential role in regulating floral development via ectopic expression in Arabidopsis thaliana and Petunia hybrida. The expression of CpAP3 is tissue-specific, with the highest level in the tepals, moderate level in carpels, and weak levels in stamen and vegetative stem tissues. Its dynamic expression during flowering is associated with flower-bud formation. Ectopic expression of CpAP3 partially rescued stamen development in ap3 mutant Arabidopsis. Although no phenotypic effect has been observed in wild-type Arabidopsis, CpAP3 overexpression in petunia brought rich morphological changes and homeotic conversions to flowers, mainly involving disruption of petal and stamen development. Expressed in a broader range than those canonical B-function regulators, the ancestral B-class gene CpAP3 can affect petal and stamen development in higher eudicots. This gene also holds some bioengineering potential in creating novel floral germplasms.

Reproductive Meristem22 is a unique marker for the early stages of stamen development

Stamens undergo a very elaborate development program that gives rise not only to many specific tissue types, but also to the male gametes. The specification of stamen identity is coordinated by a group of homeotic genes such as APETALA3 (AP3) and PISTILLATA (PI), AGAMOUS (AG) and SEPALLATA (SEP1-4) genes. Genome-wide transcriptomic comparisons between floral buds of wild-type and ap3 mutants led to the identification of the REM22 gene, which is expressed in the early stages of stamen development. This gene is member of the plant-specific B3 DNA-binding superfamily. In this work, we dissect the spatio-temporal expression pattern of REM22 during the early stages of stamen development. To this end, both in situ hybridization analyses as well as in vivo fluorescence strategies were employed. At stage 4 of flower development, REM22 is expressed exclusively in those undifferentiated cells of the floral meristem that will give rise to the stamen primordia. At stage 5, REM22 expression is restricted to the epidermal and the subepidermal layers of anther primordia. Later, this expression is confined to the middle layer and the differentiating tapetal cells. After stage 10 when all the tissues of the anther have differentiated, REM22 expression is no longer detectable. Furthermore, we examined the pREM22::GUS-GFP marker line in an inducible system where the ectopic AG function is used to promote microsporogenesis. The data support the idea that REM22 expression is a useful marker to study the early stages of stamen development.

The Rab GTPase Ypt7 is linked to retromer-mediated receptor recycling and fusion at the yeast late endosome

Organelles of the endomembrane system need to counterbalance fission and fusion events to maintain their surface-to-volume ratio. At the late mammalian endosome, the Rab GTPase Rab7 is a major regulator of fusion, whereas the homologous yeast protein Ypt7 seems to be restricted to the vacuole surface. Here, we present evidence that Ypt7 is recruited to and acts on late endosomes, where it affects multiple trafficking reactions. We show that overexpression of Ypt7 results in expansion and massive invagination of the vacuolar membrane, which requires cycling of Ypt7 between GDP- and GTP-bound states. Invaginations are blocked by ESCRT, CORVET and retromer mutants, but not by autophagy or AP-3 mutants. We also show that Ypt7-GTP specifically binds to the retromer cargo-recognition subcomplex, which--like its cargo Vps10--is found on the vacuole upon Ypt7 overproduction. Our data suggest that Ypt7 functions at the late endosome to coordinate retromer-mediated recycling with the fusion of late endosomes with vacuoles.

Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition

During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins.

The MIK region rather than the C‐terminal domain of AP3‐like class B floral homeotic proteins determines functional specificity in the development and evolution of petals

In core eudicots, euAP3-type MADS-box genes encode a PISTILLATA (PI)-derived motif, as well as a C-terminal euAP3 motif that originated from a paleoAP3 motif of an ancestral APETALA3 (AP3)-like protein through a translational frameshift mutation. To determine the functional and evolutionary relevance of these motifs, a series of point mutation and domain-swap constructs were generated, involving CsAP3, a paleoAP3-type gene from the basal angiosperm Chloranthus spicatus encoding a truncated paleoAP3 motif, and AtAP3, a euAP3-type gene from the core eudicot Arabidopsis thaliana. The chimeric constructs were expressed in A. thaliana under the control of the AP3 promoter or the CaMV 35S promoter in an ap3 mutant or wild-type background, respectively. Significant recovery of AP3 function was obtained in both complementation and ectopic expression experiments whenever the region upstream of the C-terminal motifs (MIK region) from A. thaliana was taken, even when the PI-derived motif and the truncated paleoAP3 motif of CsAP3 substituted for the corresponding sequences from AtAP3. However, no or very weak complementation or gain-of-function was seen when the MIK region was from CsAP3. Our data suggest that changes in the MIK region rather than mutations in the C-terminal domain were of crucial importance for the evolution of the functional specificity of euAP3-type proteins in stamen and petal development.

CAUT lines: a novel resource for studies of cell autonomy in Arabidopsis

Plant development is critically dependent on the interactions between clonally unrelated cell layers. The cross-talk between layers can be addressed by studies of cell autonomy. Cell autonomy is a property of genetic mosaics composed of cells of differing genotypes. Broadly, if the phenotype of a mutant tissue reflects only its genotype and is unaffected by the presence of wild-type tissue, the trait is cell-autonomous. Conversely, if the phenotype of a mutant tissue reflects that of wild-type tissue in the mosaic, the trait is non-autonomous. Here we report a novel, versatile and robust method for studies of cell autonomy in Arabidopsis. Cell autonomy (CAUT) lines consist of a collection of homozygous stocks, each containing one of 76 mapped T-DNA inserts, each of which corrects the yellow ch-42 mutant to green (CH-42) by complementation. This has the effect of translocating the colour marker to 76 new locations around the genome. X-irradiation of heterozygous CAUT line seeds results in yellow sectors, with loss of the CH-42 transgene and adjacent wild-type genes. This property can be used to remove the wild-type copy of developmental genes in appropriate heterozygotes, resulting in yellow (ch-42) sectors that are hemizygous for the trait of interest. Such sectors can provide insight into cell autonomy. Experiments using the ap1, ap3, ag and clv1 mutants show that CAUT lines are useful in the study of cell autonomy.

Virus induced gene silencing of a DEFICIENS ortholog in Nicotiana benthamiana.

Traditionally, developmental studies in plant biology have suffered from the lack of a convenient means to study gene function in non-model plant species. Here we show that virus-induced gene silencing (VIGS) is an effective new tool to study the function of orthologs of floral homeotic genes such as DEFICIENS (DEF) in non-model systems. We used a tobacco rattle virus (TRV)-based VIGS approach to study the function of the Nicotiana benthamiana DEF ortholog (NbDEF). Silencing of NbDEF in N. benthamiana using TRV-VIGS was similar to that of Antirrhinum def and Arabidopsis ap3 mutants and caused transformation of petals into sepals and stamens into carpels. Molecular analysis of the NbDEF -silenced plants revealed a dramatic reduction of the levels of NbDEF mRNA and protein in flowers. NbDEF silencing was specific and has no effect on the mRNA levels of NbTM6, the closest paralog of NbDEF. A dramatic reduction of the levels of N. benthamiana GLOBOSA (NbGLO) mRNA and protein was also observed in flowers of NbDEF-silenced plants, suggesting that cross-regulation of this GLO-like gene by NbDEF. Taken together, our results suggest that NbDEF is a functional homolog of Antirrhinum DEF. Our results are significant in that they show that TRV efficiently induces gene silencing in young and differentiating flowers and that VIGS is a promising new tool for analyses of developmental gene function in non-model organisms.

Pistillata-5, an Arabidopsis B class mutant with strong defects in petal but not in stamen development.

The Arabidopsis floral organ identity genes APETALA3 (AP3) and PISTILLATA (PI) encode related DNA-binding proteins of the MADS family. Considerable evidence supports the hypothesis that a heterodimer of AP3 and PI is an essential component of B class activity. All ap3 and pi alleles characterized to date exhibit equivalent phenotypic defects in both whorls 2 and 3. In strong ap3 and pi mutants, petals and stamens are missing and sepals and carpels develop in their place. Weak ap3 and pi mutants exhibit partial conversions of petals to sepals and stamens to carpels. In this report, we describe the isolation and characterization of pi-5, an unusual B class mutant that exhibits defects in whorl 2 where sepals develop in place of petals, but third whorl stamens are most often normal. pi-5 flowers resemble those from 35S::SEP3 antisense plants. pi-5 contains missense mutation in the K domain (PIE125K). PIE125K exhibits defects in heterodimerization with its partner protein AP3. Via a reverse yeast two-hybrid screen, AP3K139E was isolated as a compensatory mutant of PIE125K. The compensatory interaction between PIE125K and AP3K139E is observed both in yeast two-hybrid assays and in planta. On its own, AP3K139E exhibits defects in specifying both petal and stamen identity. In addition, PIE125K is defective in interaction with SEPALLATA proteins in both two- and three-hybrid assays suggesting that PIE125K is defective in forming higher order complexes of MADS proteins. The decreased concentration of PI/AP3/SEP complexes offers an explanation for the petal defects observed in both pi-5 and 35S::SEP3 antisense plants.

The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis.

The Arabidopsis floral regulatory genes APETALA3 (AP3) and PISTILLATA (PI) are required for the B function according to the ABC model for floral organ identity. AP3 and PI expression are positively regulated by the LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) genes. UFO encodes an F-box protein, and we have shown previously that UFO genetically interacts with the ASK1 gene encoding a SKP1 homologue; both the F-box containing protein and SKP1 are subunits of ubiquitin ligases. We show here that the ask1-1 mutation can enhance the floral phenotypes of weak lfy and ap3 mutants; therefore, like UFO, ASK1 also interacts with LFY and AP3 genetically. Furthermore, our results from RNA in situ hybridizations indicate that ASK1 regulates early AP3 and PI expression. These results support the idea that UFO and ASK1 together positively regulate AP3 and PI expression. We propose that the UFO and ASK1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression. Our genetic studies also indicate that ASK1 and UFO play a role in regulating the number of floral organ primordia, and we discuss possible mechanisms for such a regulation.

Molecular Characterization of the Protein Encoded by the Hermansky-Pudlak Syndrome Type 1 Gene

Hermansky-Pudlak syndrome (HPS) comprises a group of genetic disorders characterized by defective lysosome-related organelles. The most common form of HPS (HPS type 1) is caused by mutations in a gene encoding a protein with no homology to any other known protein. Here we report the identification and biochemical characterization of this gene product, termed HPS1p. Endogenous HPS1p was detected in a wide variety of human cell lines and exhibited an electrophoretic mobility corresponding to a protein of approximately 80 kDa. In contrast to previous theoretical analysis predicting that HPS1p is an integral membrane protein, we found that this protein was predominantly cytosolic, with a small amount being peripherally associated with membranes. The sedimentation coefficient of the soluble form of HPS1p was approximately 6 S as inferred from ultracentrifugation on sucrose gradients. HPS1p-deficient cells derived from patients with HPS type 1 displayed normal distribution and trafficking of the lysosomal membrane proteins, CD63 and Lamp-1. This was in contrast to cells from HPS type 2 patients, having mutations in the beta3A subunit of the AP-3 adaptor complex, which exhibited increased routing of these lysosomal proteins through the plasma membrane. Similar analyses performed on fibroblasts from 10 different mouse models of HPS revealed that only the AP-3 mutants pearl and mocha display increased trafficking of Lamp-1 through the plasma membrane. Taken together, these observations suggest that the product of the HPS1 gene is a cytosolic protein capable of associating with membranes and involved in the biogenesis and/or function of lysosome-related organelles by a mechanism distinct from that dependent on the AP-3 complex.

The yeast adaptor protein complex, AP-3, is essential for the efficient delivery of alkaline phosphatase by the alternate pathway to the vacuole.

A novel clathrin adaptor-like complex, adaptor protein (AP)-3, has recently been described in yeast and in animals. To gain insight into the role of yeast AP-3, a genetic strategy was devised to isolate gene products that are required in the absence of the AP-3 mu chain encoded by APM3. One gene identified by this synthetic lethal screen was VPS45. The Vps pathway defines the route that several proteins, including carboxypeptidase Y, take from the late Golgi to the vacuole. However, vacuolar alkaline phosphatase (ALP) is transported via an alternate, intracellular route. This suggested that the apm3-Delta vps45 synthetic phenotype could be caused by a block in both the alternate and the Vps pathways. Here we demonstrate that loss of function of the AP-3 complex results in slowed processing and missorting of ALP. ALP is no longer localized to the vacuole membrane by immunofluorescence, but is found in small punctate structures throughout the cell. This pattern is distinct from the Golgi marker Kex2p, which is unaffected in AP-3 mutants. We also show that in the apm3-Delta mutant some ALP is delivered to the vacuole by diversion into the Vps pathway. Class E vps mutants accumulate an exaggerated prevacuolar compartment containing membrane proteins on their way to the vacuole or destined for recycling to the Golgi. Surprisingly, in AP-3 class E vps double mutants these proteins reappear on the vacuole. We suggest that some AP-3-dependent cargo proteins that regulate late steps in Golgi to vacuole transport are diverted into the Vps pathway allowing completion of transfer to the vacuole in the class E vps mutant.

Role of SUPERMAN in maintaining Arabidopsis floral whorl boundaries.

The Arabidopsis gene SUPERMAN (SUP) is necessary for the proper spatial development of reproductive floral tissues. Recessive mutations cause extra stamens to form interior to the normal third whorl stamens, at the expense of fourth whorl carpel development. The mutant phenotype is associated with the ectopic expression of the B function genes, AP3 and PI, in the altered floral region, closer to the centre of the flower than in the wild type, and ap3 sup and pi sup double mutants exhibit a phenotype similar to ap3 and pi single mutants. These findings led to SUP being interpreted as an upstream negative regulator of the B function organ-identity genes, acting in the fourth whorl, to establish a boundary between stamen and carpel whorls. Here we show, using molecular cloning and analysis, that it is expressed in the third whorl and acts to maintain this boundary in developing flowers. The putative SUPERMAN protein contains one zinc-finger and a region resembling a basic leucine zipper motif, suggesting a function in transcriptional regulation.

The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens

Mutations in the APETALA3 (AP3) gene of A. thaliana result in homeotic transformations of petals to sepals and stamens to carpels. We have cloned the AP3 gene from Arabidopsis based on its homology to the homeotic flower gene deficiens (DEFA) from the distantly related plant Antirrhinum majus. The sequence of four ap3 mutant alleles and genetic mapping analysis prove that the DEFA homolog is AP3. Like several other plant homeotic genes, the AP3 gene contains a MADS box and likely acts as a transcription factor. The region-specific spatial expression pattern of AP3 rules out certain types of sequential models of flower development and argues in favor of a spatial model based on positional information. Since DEFA and AP3 have very similar protein products, mutant phenotypes, and spatial expression patterns, it is likely that these genes are cognate homologs.