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.     

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.     

The Drosophila melanogaster retinophilin gene encodes the peripheral membrane protein in photoreceptor cells

The Drosophila melanogaster retinophilin (rtp) gene encoding the protein containing MORN (Membrane Occupation and Recognition Nexus) motifs that are known to interact with the plasma membrane has been identified to be expressed predominantly in adult eyes by several independent studies. Isolation and characterization of rtp mutant flies showed that the gene is involved in the phototransduction process by interacting with NINAC (neither inactivation nor afterpotential C) protein in adult photoreceptor cells. The gene was also reported to be involved in phagocytosis in embryos. We examined rtp gene expression during D. melanogaster development and in adult head tissues. The results showed that the gene is expressed at detectable levels only in adult photoreceptor cells but not in other developmental stages and other adult tissues, confirming its phototransduction functions. The RTP protein contains only 4 MORN motifs, whereas 8 MORN motifs are reported to be required for interactions with the plasma membrane. We found that RTP protein is present free in the cytosol and also is bound peripherally to the plasma membrane; this binding ability was found to be modulated by light. Our results suggest that the D. melanogaster RTP protein is a light-regulated peripheral membrane protein of photoreceptor cells.     

Trypanosomatid Pin1‐Type Peptidyl‐Prolyl Isomerase Is Cytosolic and Not Essential for Cell Proliferation

Pin1‐type peptidyl‐prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)‐Pro residues, regulating various cellular events. Previously, we reported a Pin1‐type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1‐like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi‐mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two‐hybrid assay, we identified a MORN domain‐containing protein as putative Pin1‐binding partners. These data suggest that Pin1‐mediated signaling mechanism plays a different role in protozoan parasites.     

Structure and evolution of ENTH and VHS/ENTH‐like domains in tepsin

Tepsin is currently the only accessory trafficking protein identified in adaptor‐related protein 4 (AP4)‐coated vesicles originating at the trans‐Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C‐terminus have been characterized, but the biological role of tepsin remains unknown. We determined X‐ray crystal structures of the tepsin epsin N‐terminal homology (ENTH) and VHS/ENTH‐like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8 and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine‐based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure.      

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1‐GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1‐GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R≈35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1‐GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1‐GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1‐GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.     

Structural insight into the interaction of proteins containing NPF,DPF, and GPF motifs with the C‐terminal EH‐domain of EHD1

Eps15 homology (EH)‐domain containing proteins are regulators of endocytic membrane trafficking. EH‐domain binding to proteins containing the tripeptide NPF has been well characterized, but recent studies have shown that EH‐domains are also able to interact with ligands containing DPF or GPF motifs. We demonstrate that the three motifs interact in a similar way with the EH‐domain of EHD1, with the NPF motif having the highest affinity due to the presence of an intermolecular hydrogen bond. The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues.     

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.     

Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc‐binding sites in the C‐terminal domain

Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N‐terminal catalytic domain and a 30 kDa C‐terminal zinc‐binding region (ZD domain) which has three adjacent tetra‐cysteine zinc‐binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zinc‐binding motifs in ZD domain and both the first and second zinc‐binding motifs are required for iron binding in TopA. The site‐directed mutagenesis studies further reveal that while the mutation of the third zinc‐binding motif has very little effect on TopA's activity, mutation of the first two zinc‐binding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo, indicating that the first two zinc‐binding motifs in TopA are crucial for its function. The DNA‐binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single‐stranded (ss) DNA‐binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity.     

MotifAnalyzer‐PDZ: A computational program to investigate the evolution of PDZ‐binding target specificity

Recognition of short linear motifs (SLiMs) or peptides by proteins is an important component of many cellular processes. However, due to limited and degenerate binding motifs, prediction of cellular targets is challenging. In addition, many of these interactions are transient and of relatively low affinity. Here, we focus on one of the largest families of SLiM‐binding domains in the human proteome, the PDZ domain. These domains bind the extreme C‐terminus of target proteins, and are involved in many signaling and trafficking pathways. To predict endogenous targets of PDZ domains, we developed MotifAnalyzer‐PDZ, a program that filters and compares all motif‐satisfying sequences in any publicly available proteome. This approach enables us to determine possible PDZ binding targets in humans and other organisms. Using this program, we predicted and biochemically tested novel human PDZ targets by looking for strong sequence conservation in evolution. We also identified three C‐terminal sequences in choanoflagellates that bind a choanoflagellate PDZ domain, the Monsiga brevicollis SHANK1 PDZ domain (mbSHANK1), with endogenously‐relevant affinities, despite a lack of conservation with the targets of a homologous human PDZ domain, SHANK1. All three are predicted to be signaling proteins, with strong sequence homology to cytosolic and receptor tyrosine kinases. Finally, we analyzed and compared the positional amino acid enrichments in PDZ motif‐satisfying sequences from over a dozen organisms. Overall, MotifAnalyzer‐PDZ is a versatile program to investigate potential PDZ interactions. This proof‐of‐concept work is poised to enable similar types of analyses for other SLiM‐binding domains (e.g., MotifAnalyzer‐Kinase). MotifAnalyzer‐PDZ is available at http://motifAnalyzerPDZ.cs.wwu.edu .     

Linking 3D and 2D binding kinetics of membrane proteins by multiscale simulations

Membrane proteins are among the most functionally important proteins in cells. Unlike soluble proteins, they only possess two translational degrees of freedom on cell surfaces, and experience significant constraints on their rotations. As a result, it is currently challenging to characterize the in situ binding of membrane proteins. Using the membrane receptors CD2 and CD58 as a testing system, we developed a multiscale simulation framework to study the differences of protein binding kinetics between 3D and 2D environments. The association and dissociation processes were implemented by a coarse‐grained Monte‐Carlo algorithm, while the dynamic properties of proteins diffusing on lipid bilayer were captured from all‐atom molecular dynamic simulations. Our simulations show that molecular diffusion, linker flexibility and membrane fluctuations are important factors in adjusting binding kinetics. Moreover, by calibrating simulation parameters to the measurements of 3D binding, we derived the 2D binding constant which is quantitatively consistent with the experimental data, indicating that the method is able to capture the difference between 3D and 2D binding environments. Finally, we found that the 2D dissociation between CD2 and CD58 is about 100‐fold slower than the 3D dissociation. In summary, our simulation framework offered a generic approach to study binding mechanisms of membrane proteins.     

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.     

Signalling in ciliates: long‐ and short‐range signals and molecular determinants for cellular dynamics

In ciliates, unicellular representatives of the bikont branch of evolution, inter‐ and intracellular signalling pathways have been analysed mainly in Paramecium tetraurelia, Paramecium multimicronucleatum and Tetrahymena thermophila and in part also in Euplotes raikovi. Electrophysiology of ciliary activity in Paramecium spp. is a most successful example. Established signalling mechanisms include plasmalemmal ion channels, recently established intracellular Ca²⁺‐release channels, as well as signalling by cyclic nucleotides and Ca²⁺. Ca²⁺‐binding proteins (calmodulin, centrin) and Ca²⁺‐activated enzymes (kinases, phosphatases) are involved. Many organelles are endowed with specific molecules cooperating in signalling for intracellular transport and targeted delivery. Among them are recently specified soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), monomeric GTPases, H⁺‐ATPase/pump, actin, etc. Little specification is available for some key signal transducers including mechanosensitive Ca²⁺‐channels, exocyst complexes and Ca²⁺‐sensor proteins for vesicle–vesicle/membrane interactions. The existence of heterotrimeric G‐proteins and of G‐protein‐coupled receptors is still under considerable debate. Serine/threonine kinases dominate by far over tyrosine kinases (some predicted by phosphoproteomic analyses). Besides short‐range signalling, long‐range signalling also exists, e.g. as firmly installed microtubular transport rails within epigenetically determined patterns, thus facilitating targeted vesicle delivery. By envisaging widely different phenomena of signalling and subcellular dynamics, it will be shown (i) that important pathways of signalling and cellular dynamics are established already in ciliates, (ii) that some mechanisms diverge from higher eukaryotes and (iii) that considerable uncertainties still exist about some essential aspects of signalling.     

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.     

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

Vacuolar H⁺‐ATPase (V‐ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V‐ATPase activity is regulated by reversible disassembly, resulting in cytosolic V₁‐ATPase and membrane‐integral V₀ proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein‐protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein–protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc‐reconstituted V‐ATPase (V₁V₀ND). We show that V₁V₀ND can be immobilized on streptavidin‐coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.     

Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.