Invertebrate and Vertebrate Class III Myosins Interact with MORN Repeat-Containing Adaptor Proteins

In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior.     

<Emphasis Type="Italic">Drosophila</Emphasis> retinophilin contains MORN repeats and is conserved in humans

The function of conserved novel human genes can be efficiently addressed in genetic model organisms. From a collection of genes expressed in the Drosophila visual system, cDNAs expressed in vertebrates were identified and one similar to a novel human gene was chosen for further investigation. The results reported here characterize the Drosophila retinophilin gene and demonstrate that a similar gene is expressed in the human retina. The Drosophila and human retinophilin sequences are 50% identical, and they share an additional 16% conserved substitutions. Examination of the cDNA and genomic sequence indicates that it corresponds to the gene CG10233 of the annotated genome and predicts a 22.7 kDa protein. Polyclonal antibodies generated to a predicted retinophilin peptide recognize an antigen in Drosophila photoreceptor cells. The retinophilins encode 4 copies of a repeat associated with a Membrane Occupation and Recognition Nexus (MORN) function first discovered in junctophilins, which may interact with the plasma membrane. These results therefore show that Drosophila retinophilin is expressed in fly photoreceptor cells, demonstrate that a conserved human gene is expressed in human retina, and suggest that a mutational analysis of the Drosophila gene would be valuable.     

Tetrahymena Expresses More than a Hundred Proteins with Lipid‐binding MORN Motifs that can Differ in their Subcellular Localisations

Proteins with membrane occupation and recognition nexus (MORN) motifs are associated with cell fission in apicomplexan parasites, chloroplast division in Arabidopsis and the motility of sperm cells. We found that ciliates are among those that encode the largest variety of MORN proteins. Tetrahymena thermophila expresses 129 MORN protein‐encoding genes, some of which are specifically up‐regulated during conjugation. A lipid‐binding assay underpins the assumption that the predominant function of MORN motifs themselves is to confer the ability of lipid binding. The localisation of four MORN candidate proteins with similar characteristics highlights the functional diversity of this group especially in ciliates.     

Overexpression of BrMORN, a novel ‘membrane occupation and recognition nexus’ motif protein gene from Chinese cabbage, promotes vegetative growth and seed production in Arabidopsis

Proteins that contain membrane occupation and recognition nexus (MORN) motifs regulate various aspects of cellular metabolism by localizing proteins in different cellular organelles. The full-length Brassica rapa MORN motif protein (BrMORN) cDNA consists of 1,510 bp encoding 502 deduced amino acids with a predicted molecular mass of 55.8 kDa and an isoelectric point of 9.72. BrMORN is a novel protein composed of two N-terminal transmembrane helices and seven C-terminal MORN motifs and it appears to be localized on the plastid envelope. BrMORN expression was relatively high in actively-growing tissues, but low in mature tissues and under some abiotic stresses. Arabidopsis thaliana plants overexpressing BrMORN showed an enhanced rate of growth, hypocotyl elongation, and increases in the size of vegetative organs and seed productivity under normal growth conditions. In addition, cell size in Arabidopsis plants overexpressing BrMORN was 24% larger than that of wild-type plants, implying that the increase in the size of vegetative organs is due to cell enlargement. The increased size of the vegetative organs also led to increased seed production. Our data suggest that the MORN motif of BrMORN may act at the plastid envelope and facilitate plant growth via cell enlargement.     

Isolation of mRNA from specific tissues of Drosophila by mRNA tagging

To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes.     

The N-terminal membrane occupation and recognition nexus domain of Arabidopsis phosphatidylinositol phosphate kinase 1 regulates enzyme activity

The type I B family of phosphatidylinositol phosphate kinases (PIPKs) contain a characteristic region of Membrane Occupation and Recognition Nexus (MORN) motifs at the N terminus. These MORN motifs are not found in PIPKs from other eukaryotes. To understand the impact of the additional N-terminal domain on protein function and subcellular distribution, we expressed truncated and full-length versions of AtPIPK1, one member of this family of PIPKs, in Escherichia coli and in tobacco cells grown in suspension culture. Deletion of the N-terminal MORN domain (amino acids 1-251) of AtPIPK1 increased the specific activity of the remaining C-terminal peptide (DeltaMORN) >4-fold and eliminated activation by phosphatidic acid (PtdOH). PtdOH activation could also be eliminated by mutating Pro(396) to Ala (P396A) in the predicted linker region between the MORN and the kinase homology domains. AtPIPK1 is product-activated and the MORN domain binds PtdIns(4,5)P(2). Adding back the MORN peptide to DeltaMORN or to the PtdOH-activated full-length protein increased activity approximately 2-fold. Furthermore, expressing the MORN domain in vivo increased the plasma membrane PtdInsP kinase activity. When cells were exposed to hyperosmotic stress, the MORN peptide redistributed from the plasma membrane to a lower phase or endomembrane fraction. In addition, endogenous PtdInsP kinase activity increased in the endomembrane fraction of hyperosmotically stressed cells. We conclude that the MORN peptide can regulate both the function and distribution of the enzyme in a manner that is sensitive to the lipid environment.     

Cross species analysis of Prominin reveals a conserved cellular role in invertebrate and vertebrate photoreceptor cells

The two fundamental types of photoreceptor cells have evolved unique structures to expand the apical membrane to accommodate the phototransduction machinery, exemplified by the cilia-based outer segment of the vertebrate photoreceptor cell and the microvilli-based rhabdomere of the invertebrate photoreceptor. The morphogenesis of these compartments is integral for photoreceptor cell integrity and function. However, little is known about the elementary cellular and molecular mechanisms required to generate these compartments. Here we investigate whether a conserved cellular mechanism exists to create the phototransduction compartments by examining the functional role of a photoreceptor protein common to both rhabdomeric and ciliated photoreceptor cells, Prominin. First and foremost we demonstrate that the physiological role of Prominin is conserved between rhabdomeric and ciliated photoreceptor cells. Human Prominin1 is not only capable of rescuing the corresponding rhabdomeric Drosophila prominin mutation but also demonstrates a conserved genetic interaction with a second photoreceptor protein Eyes Shut. Furthermore, we demonstrate the Prominin homologs in vertebrate and invertebrate photoreceptors require the same structural features and post-translational modifications for function. Moreover, expression of mutant human Prominin1, associated with autosomal dominant retinal degeneration, in rhabdomeric photoreceptor cells disrupts morphogenesis in ways paralleling retinal degeneration seen in ciliated photoreceptors. Taken together, our results suggest the existence of an ancestral Prominin-directed cellular mechanism to create and model the apical membranes of the two fundamental types of photoreceptor cells into their respective phototransduction compartments.     

Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.     

MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding

Multiple repeats of membrane occupation and recognition nexus （MORN） motifs were detected in plant phosphatidylinositl monophosphate kinase （PIPK）, a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs （with varied numbers at ranges of 7-9）, which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an -100 amino-acid ＂island＂ region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET （sequence of membrane-targeting） system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK 1, together with the 104 amino-acid ＂island＂ region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI（4,5）P2. These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.     

Membrane maintenance and electrical properties of photoreceptors of wild-type andrpa (receptor potential absent) mutant blowflies (Calliphora erythrocephala)

Summary The maintenance of photoreceptor cell membranes in the blowfly was investigated in relation to the diurnal cycle, age, and therpa (receptor potential absent) phototransduction mutation. The effect of disturbed membrane assembly on the electrical membrane properties was examined using single-electrode discontinuous current-clamp techniques. In wild-type flies the cross-sectional dimensions of the rhabdomeres were markedly reduced with age, and the quantity of synthetic organelles decreased concurrently, whereas no correlation was found between the diurnal cycle and membrane turnover. Therpa mutation is thought to block the visual transduction cascade in photoreceptor cells and to lead to degeneration of the photoreceptor cell bodies. The volume of rhabdomeres decreased markedly inrpa mutants and the quantity of synthetic organelles was reduced significantly, indicating an imbalance between photoreceptive membrane renewal and degradation. Also, the plasma membrane underwent degenerative changes. The passive electrical properties of photoreceptor cells — resting membrane voltages and input resistances — were only slightly changed from those of wild-type flies, although the photoreceptive membrane did not depolarize in response to light. This indicates no apparent disturbance in the function of the ionic channels in these membranes. Taken together, these results suggest that the photoreceptor cells need a functional phototransduction cascade with its feedback controls to maintain continuous renewal of rhabdomeres, but that the plasma membrane maintains its normal electrochemical properties despite extreme morphological degeneration of photoreceptor cell.     

Conservation of copper-transporting P(IB)-type ATPase function

Copper-transporting P(IB)-type ATPases are highly conserved, and while unicellular eukaryotes and invertebrates have only one, a gene duplication has occurred during vertebrate evolution. Copper-induced trafficking of mammalian ATP7A and ATP7B from the trans-Golgi Network towards the plasma membrane is critical for their role in copper homeostasis. In polarized epithelial cells ATP7A and ATP7B traffic towards the basolateral and apical membranes respectively. We examined the localization and function of DmATP7, the single Drosophila melanogaster orthologue, in cultured D. melanogaster and mammalian cells to explore the conservation of P(IB)-type ATPase function. Comparative genomic analysis demonstrated motifs involved in basolateral targeting and retention of ATP7A were conserved in DmATP7, whereas ATP7B targeting motifs were not. DmATP7 expression was able to correct the copper hyper-accumulation phenotype of cultured fibroblasts from a Menkes disease patient expressing a null ATP7A allele. DmATP7 was able to transport copper to the cupro-enzyme tyrosinase and under elevated copper conditions DmATP7 was able to traffic towards the plasma membrane and efflux copper, essentially phenocopying ATP7A. When expressed in polarized Madin-Darby Canine Kidney cells, DmATP7 translocated towards the basolateral membrane when exposed to elevated copper, similar to ATP7A. These results demonstrate DmATP7 is able to functionally compensate for the absence of ATP7A, with important trafficking motifs conserved in these distantly related orthologues.     

Nucleotide sequence of a Drosophila melanogaster cDNA encoding a calnexin homologue

A cDNA which encodes a calnexin (Cnx)-like protein from Drosophila melanogaster has been characterized. The deduced amino acid sequence shares several regions of homology with Cnx from other sources with two conserved motifs each repeated four times. The gene was found to be transcribed in various tissues and at all developmental stages. We have mapped the gene at chromosomal position 99A and we have also mapped the related gene coding for Drosophila calreticulin at 85E.     

Maintenance of Rhodopsin levels in Drosophila photoreceptor and phototransduction requires Protein Kinase D

ABSTRACT

During Drosophila phototransduction, the G protein coupled receptor (GPCR) Rhodopsin (Rh1) transduces photon absorption into electrical signal via G-protein coupled activation of phospholipase C (PLC). Rh1 levels in the plasma membrane are critical for normal sensitivity to light. In this study, we report that Protein Kinase D (dPKD) regulates Rh1 homeostasis in adult photoreceptors. Although eye development and retinal structure are unaffected in the dPKD hypomorph (dPKD^H), it exhibited elevated levels of Rh1. Surprisingly, despite having elevated levels of Rh1, no defect was observed in the electrical response to light in these flies. By contrast the levels of another transmembrane protein of the photoreceptor plasma membrane, Transient receptor potential (TRP) was not altered in dPKD^H. Our results indicate that dPKD is dispensable for eye development but is required for maintaining Rh1 levels in adult photoreceptors.     

Identification and characterization of a new 34 kDa MORN motif-containing sporozoite surface-exposed protein,Cp-P34, unique to Cryptosporidium

Despite the public health impact of childhood diarrhea caused by Cryptosporidium, effective drugs and vaccines against this parasite are unavailable. Efforts to identify vaccine targets have focused on critical externally exposed virulence factors expressed in the parasite s invasive stages. However, no single surface antigen has yet been found that can elicit a significant protective immune response and it is likely that pooling multiple immune targets will be necessary. Discovery of surface proteins on Cryptosporidium sporozoites is therefore vital to this effort to develop a multi-antigenic vaccine. In this study we applied a novel single-domain camelid antibody (VHH) selection method to identify immunogenic proteins expressed on the surface of Cryptosporidium parvum sporozoites. By this approach, VHHs were identified that recognize two sporozoite surface-exposed antigens, the previously identified gp900 and an unrecognized immunogenic protein, Cp-P34. This Cp-P34 antigen, which contains multiple Membrane Occupation and Recognition Nexus (MORN) repeats, is found in excysted sporozoites as well as in the parasite s intracellular stages. Cp-P34 appears to accumulate inside the parasite and transiently appears on the surface of sporozoites to be shed in trails. Identical or nearly identical orthologs of Cp-P34 are found in the Cryptosporidium hominis and Cryptosporidium tyzzeri genomes. Except for the conserved MORN motifs, the Cp-P34 gene shares no significant homology with genes of other protozoans and thus appears to be unique to Cryptosporidium spp. Cp-P34 elicits immune responses in naturally exposed alpacas and warrants further investigation as a potential vaccine candidate.     

Golgi-localized MORN1 promotes lipid droplet abundance and enhances tolerance to multiple stresses in Arabidopsis

Lipid droplet (LD) in vegetative tissues has recently been implicated in environmental responses in plants, but its regulation and its function in stress tolerance are not well understood. Here, we identified a Membrane Occupation and Recognition Nexus 1 (MORN1) gene as a contributor to natural variations of stress tolerance through genome-wide association study in Arabidopsis thaliana. Characterization of its loss-of-function mutant and natural variants revealed that the MORN1 gene is a positive regulator of plant growth, disease resistance, cold tolerance, and heat tolerance. The MORN1 protein is associated with the Golgi and is also partly associated with LD. Protein truncations that disrupt these associations abolished the biological function of the MORN1 protein. Furthermore, the MORN1 gene is a positive regulator of LD abundance, and its role in LD number regulation and stress tolerance is highly linked. Therefore, this study identifies MORN1 as a positive regulator of LD abundance and a contributor to natural variations of stress tolerance. It implicates a potential involvement of Golgi in LD biogenesis and strongly suggests a contribution of LD to diverse processes of plant growth and stress responses.     

uninflatable encodes a novel ectodermal apical surface protein required for tracheal inflation in Drosophila

The tracheal system of Drosophila melanogaster has proven to be an excellent model system for studying the development of branched tubular organs. Mechanisms regulating the patterning and initial maturation of the tracheal system have been largely worked out, yet important questions remain regarding how the mature tubes inflate with air at the end of embryogenesis, and how the tracheal system grows in response to the oxygen needs of a developing larva that increases nearly 1000-fold in volume over a four day period. Here we describe the cloning and characterization of uninflatable (uif), a gene that encodes a large transmembrane protein containing carbohydrate binding and cell signaling motifs in its extracellular domain. Uif is highly conserved in insect species, but does not appear to have a true ortholog in vertebrate species. uif is expressed zygotically beginning in stage 5 embryos, and Uif protein localizes to the apical plasma membrane in all ectodermally derived epithelia, most notably in the tracheal system. uif mutant animals show defects in tracheal inflation at the end of embryogenesis, and die primarily as larvae. Tracheal tubes in mutant larvae are often crushed or twisted, although tracheal patterning and maturation appear normal during embryogenesis. uif mutant larvae also show defects in tracheal growth and molting of their tracheal cuticle.     

A Novel Little Membrane Protein Confers Salt Tolerance in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plasma membrane proteins play critical roles in sensing and responding abiotic and biotic stresses in plants. In the present study, we characterized a previously unknown gene stress associated little protein 1 (SALP1) encoding a plasma membrane protein. SALP1, a small and plant-specific membrane protein, contains only 74 amino acid residues. SALP1 was constitutively expressed in various rice tissues while highly expressed in roots, leaf blade, and immature panicles. Expression analysis indicated that SALP1 was induced by various abiotic stresses and abscisic acid (ABA). Subcellular localization assay indicated that SALP1 was localized on plasma membrane in rice protoplast cells. Overexpressing of SALP1 in rice improved salt tolerance through increasing free proline contents and the expression level of OsP5CS gene, and balancing ion contents under salt stress. Moreover, SALP1 transgenic rice showed reduced sensitivity to ABA treatment, and expression level of SALP1 is not altered by ABI5-like 1 protein. Conclusively, SALP1, a novel membrane protein, is involved in salt tolerance through an ABA-independent signaling pathway in rice.     

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.