Retromer-Mediated Protein Sorting and Vesicular Trafficking

Retromer is an evolutionarily conserved multimeric protein complex that mediates intracellular transport of various vesicular cargoes and functions in a wide variety of cellular processes including polarized trafficking,developmental signaling and lysosome biogenesis.Through its interaction with the Rab GTPases and their effectors,membrane lipids,molecular motors,the endocytic machinery and actin nucleation promoting factors,retromer regulates sorting and trafficking of transmembrane proteins from endosomes to the trans-Golgi network(TGN) and the plasma membrane.In this review.I highlight recent progress in the understanding of relromer-medialed protein sorting and vesicle trafficking and discuss how retromer contributes to a diverse set of developmental,physiological and pathological processes.     

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcll is male sterile as a result of the failure ofpollen germination. We show that the bcll mutant allele cannot be transmitted by male gametophytes and no homozygous bcll mutants were obtained. Analysis of pollen developmental stages indicates that the bcll mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting （VPS） in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.     

Arabidopsis shaker pollen inward K~+ channel SPIK functions in SnRK1 complex-regulated pollen hydration on the stigma

Pollen hydration is a critical step that determines pollen germination on the stigma. KINβγ is a plantspecific subunit of the SNF1-related protein kinase 1 complex(SnRK1 complex). In pollen of the Arabidopsis kinbg mutant, the levels of reactive oxygen species were decreased which lead to compromised hydration of the mutant pollen on the stigma. In this study, we analyzed gene expression in kinβγ mutant pollen by RNA-seq and found the expression of inward shaker K~+ channel SPIK was down-regulated in the kinβγ pollen. Furthermore, we showed that the pollen hydration of the Arabidopsis spik mutant was defective on the wild-type stigma, although the mutant pollen demonstrated normal hydration in vitro.Additionally, the defective hydration of spik mutant pollen could not be rescued by the wild-type pollen on the stigma,indicating that the spik mutation deprived the capability of pollen absorption on the stigma. Our results suggest that the Arabidopsis SnRK1 complex regulates SPIK expression,which functions in determining pollen hydration on the stigma.     

Cell polarity: Regulators and mechanisms in plants

Cell polarity plays an important role in a wide range of biological processes in plant growth and development.Cell polarity is manifested as the asymmetric distribution of molecules,for example,proteins and lipids,at the plasma membrane and inside of a cell.Here,we summarize a few polarized proteins that have been characterized in plants and we review recent advances towards understanding the molecular mechanism for them to polarize at the plasma membrane.Multiple mechanisms,including membrane trafficking,cytoskeletal activities,and protein phosphorylation,and so forth define the polarized plasma membrane domains.Recent discoveries suggest that the polar positioning of the proteo-lipid membrane domain may instruct the formation of polarity complexes in plants.In this review,we highlight the factors and regulators for their functions in establishing the membrane asymmetries in plant development.Furthermore,we discuss a few outstanding questions to be addressed to better understand the mechanisms by which cell polarity is regulated in plants.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.     

Modulation of cell surface GABA(B) receptors by desensitization, trafficking and regulated degradation

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.     

14-3-3 λ protein interacts with ADF1 to regulate actin cytoskeleton dynamics in Arabidopsis

Actin cytoskeleton dynamics is critical for variety of cellular events including cell elongation, division and morphogenesis, and is tightly regulated by numerous groups of actin binding proteins. However it is not well understood how these actin binding proteins are modulated in a physiological condition by their interaction proteins. In this study, we describe that Arabidopsis 14-3-3 λ protein interacted with actin depolymerizing factor 1(ADF1) in plant to regulate F-actin stability and dynamics. Loss of 14-3-3 λin Arabidopsis resulted in longer etiolated hypocotyls in dark and changed actin cytoskeleton architecture in hypocotyl cells. Overexpression of ADF1 repressed 14-3-3 λ mutant hypocotyl elongation and actin dynamic phenotype. In addition, the phosphorylation level of ADF1 was increased and the subcellular localization of ADF1 was altered in 14-3-3 λ mutant. Consistent with these observations, the actin filaments were more stable in 14-3-3 λ mutant. Our results indicate that 14-3-3 λ protein mediates F-actin dynamics possibly through inhibiting ADF1 phosphorylation in vivo.     

IQ67 DOMAIN protein 21 is critical for indentation formation in pavement cell morphogenesis

In plants, cortical microtubules anchor to the plasma membrane in arrays and play important roles in cell shape. However, the molecular mechanism of microtubule binding proteins, which connect the plasma membrane and cortical microtubules in cell morphology remains largely unknown. Here, we report that a plasma membrane and microtubule duallocalized IQ67 domain protein, IQD21, is critical for cotyledon pavement cell(PC) morphogenesis in Arabidopsis. iqd21 mutation caused increased indentation wi...     

Intracellular NHX-Type Cation/H＋ Antiporters inPlants

Cells depend on the homeostatic maintenance of pHwithin specific cellular compartments to ensure optimalconditions for metabolic and enzymatic processes as wellas protein structure and function. In the animal secre-tory pathway, cells maintain distinct luminal pHs withinvarious compartments （Paroutis et al., 2004）. Among themany molecular players that contribute to pH and ionhomeostasis in plants, Na＋（K＋）/H＋ exchangers （also knownas NHX-type cation/H＋ antiporters） appear to be particu-larly important for the regulation of a wide variety ofphysiological processes, including cell expansion, cellvolume regulation, osmotic adjustment, pH regulation,membrane trafficking, protein processing, and cellularstress responses （Pardo et al., 2006; Rodriguez-Rosaleset al., 2009; Bassil et al., 2012）. In plants, NHX antiportersappeared early in evolution and are ubiquitously encodedmembers of the CPA1 cation/H＋ antiporters subgroupthat belongs to the large family of monovalent cation/H＋ transporters CPA （Brett et al., 2005）. NHX antiport-ers are found, thus far, in all sequenced plant genomes（Bassil et al., 2012; Chanroj et al., 2012）. In Arabidopsis,the NHX family consists of eight isoforms, six of whichare intracellular （AtNHXl-AtNHX6）, located either to thevacuole （AtNHXl to AtNHX4） or endosomes （AtNHX5 andAtNHX6） and an additional two more divergent members（AtNHX7/SOSl and AtNHX8） at the plasma membrane（Bassil et al., 2012）. Orthologous sequences in each of thethree classes （plasma membrane, vacuolar, or endosomal）appear in all sequenced genomes, suggesting that distinctfunctional NHX classes appeared early in evolution andmay have conserved roles that are compartment-specific（Bassil et al., 2012）. Emerging new evidence highlightsthe importance of particular intracellular NHX antiport-ers in the regulation of vesicular and vacuolar pH andK＋ homeostasis. Vacuolar NHXs are needed to maintainK＋ homeostasis between the vacuole and cytosol, with-out which cell expansion is compromised （Bassil et al.,2011b）. Other NHX isoforms （endosomal） are requiredfor membrane trafficking and raise interesting new ques-tions about the role of pH and ion homeostasis in proteinprocessing and trafficking in the endomembrane system（Bassil et al., 2011a）. In this update, we aim to highlightrecent new evidence on intracellular NHX antiportersand emphasize possible novel and important cellular pro-cesses regulated by this particularly interesting group oftransporters.     

Requirement of KNAT1/BP for the Development of Abscission Zones in Arabidopsis thaliana

The KNAT1 gene is a member of the Class I KNOXhomeobox gene family and is thought to play an important role in meristem development and leaf morphogenesis. Recent studies have demonstrated that KNAT1/BP regulates the architecture of the inflorescence by affecting pedicle development in Arabidopsis thaliana. Herein, we report the characterization of an Arabidopsis T-DNA insertion mutant that shares considerable phenotypic similarity to the previously identified mutant brevipedicle （bp）. Molecular and genetic analyses showed that the mutant is allelic to bp and that the T-DNA is located within the first helix of the KNAT1 homeodomain （HD）. Although the mutation causes a typical abnormality of short pedicles, propendent siliques, and semidwarfism, no obvious defects are observed in the vegetative stage. A study on cell morphology showed that asymmetrical division and inhibition of cell elongation contribute to the downward-pointing and shorter pedicle phenotype. Loss of KNAT/BPfunction results in the abnormal development of abscission zones. Mlcroarray analysis of gene expression profiling suggests that KNAT1/BP may regulate abscission zone development through hormone signaling and hormone metabolism in Arabidopsis.     

Bagging with CTD- A Novel Signature for the Hierarchical Prediction of Secreted Protein Trafficking in Eukaryotes

Protein trafficking or protein sorting in eukaryotes is a complicated process and is carried out based on the information contaified in the protein. Many methods reported prediction of the subcellular location of proteins from sequence information. However, most of these prediction methods use a flat structure or parallel architecture to perform prediction. In this work, we introduce ensemble classifiers with features that are extracted directly from full length protein sequences to predict locations in the protein-sorting pathway hierarchically. Sequence driven features, sequence mapped features and sequence autocorrelation features were tested with ensemble learners and their performances were compared. When evaluated by independent data testing, ensemble based-bagging algorithms with sequence feature composition, transition and distribution （CTD） successfully classified two datasets with accuracies greater than 90%. We compared our results with similar published methods, and our method equally performed with the others at two levels in the secreted pathway. This study shows that the feature CTD extracted from protein sequences is effective in capturing biological features among compartments in secreted pathways.     

In vivo Studies on the Roles of Tic55-Related Proteins in Chloroplast Protein Import in Arabidopsis thaliana

The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis： atTic55-11 and AtPTC52 （Protochlorophyllide-dependent Trans- Iocon Component, 52 kDa; has also been called atTic55-1V）. Our phylogenetic analysis shows that attic55-11 is an ortholog of psTic55 from pea （Pisum sativurn）, and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild- type： visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-11 mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-11 and AtPTC52 are not strictly required for functional protein import in Arabidopsis.     

Arabidopsis OBG-Like GTPase （AtOBGL）Is Localized in Chloroplasts and Has an Essential Function in Embryo Development

OBG-like GTPases, a subfamily of P-loop GTPases, have divers and important functions in bacteria, including initiation of sporulation, DNA replication, and protein translation. Homologs of the Bacillus subtilis spo0B GTP-binding protein （OBG） can be found in plants and algae but their specific function in these organisms has not yet been elucidated. Here, it is shown that ATSG18570 encodes an Arabidopsis thaliana OBG-like protein （AtOBGL） that is localized in chlor- oplasts. In contrast to the bacterial members of this protein family, AtOBGL and other OBG-like proteins from green algae and plants possess an additional N-terminal domain, indicating functional adaptation. Disruption of the gene locus of ATOBGL by TDNA insertion resulted in an embryo-lethal phenotype and light microscopy using Normarski optics revealed that embryo maturation in the atobgl mutant is arrested at the late globular stage before development of a green embryo. Expression of 35S：：ATOBGL within the atobgl mutant background could rescue the mutant phenotype, confirming that embryo-lethality is caused by the loss of AtOBGL. Together, the data show that the bacterial-derived OBG-like GTPases have retained an essential role in chloroplasts of plants and algae. They furthermore corroborate the significance of chloroplast functions for embryo development -- an important stage within the Arabidopsis lifecycle.     

A Crucial Role of the RGS Domain in Trans- Golgi Network Export of AtRGS1 in the Protein Secretory Pathway

The secretory pathway is responsible for the transport of newly synthesized transmembrane proteins from the endoplasmic reticulum to their destinations via the Golgi/trans-Golgi network （TGN）, Cargo proteins at each sta- tion are actively sorted by specific sorting signals on the cargo and the corresponding coat complexes. Here, we used the Arabidopsis regulator of G-protein signaling （AtRGS1）, which contains an N-terminal potentially sensing glucose seven-transmembrane domain and a C-terminal RGS domain, as a model to uncover sorting motifs required for its cell surface expression. Expression of wild-type and truncated or mutated AtRGS1 fluorescent fusion proteins identified two cysteine residues in the extracellular N-terminus that are essential for endoplasmic reticulum exit and/or correct folding of AtRGS1. The linker between the seven-transmembrane and RGS domains contains an endoplasmic reticulum export signal, whereas the C-terminus is dispensable for the plasma membrane expression of AtRGS1. Interestingly, deletion of the RGS domain results in Golgi/TGN localization of the truncated AtRGS1. Further analysis using site-directed mutagen- esis showed that a tyrosine-based motif embedded in the RGS domain is essential for Golgi/TGN export of AtRGS1. These results reveal a new role for the RGS domain in regulating AtRGS1 trafficking from the Golgi/TGN to the plasma membrane and explain the interaction between the seven-transmembrane and RGS domains.     

Plant and yeast NHX antiporters: roles in membrane trafficking

The plant NHX gene family encodes Na + /H + antiporters which are crucial for salt tolerance, potassium homeostasis and cellular pH regulation. Understanding the role of NHX antiporters in membrane trafficking is becoming an increasingly interesting subject of study. Membrane trafficking is a central cellular process during which proteins, lipids and polysaccharides are continuously exchanged among membrane compartments. Yeast ScNhx1p, a prevacuole/ vacuolar Na + /H + antiporter, plays an important role in regulating pH to control trafficking out of the endosome. Evidence begins to accumulate that plant NHX antiporters might function in regulating membrane trafficking in plants.     

Phosphatidic Acid （PA） PP2A Activity and PIN1 Binds PP2AA1 to Regulate Polar Localization

Phospholipase D （PLD） exerts broad biological functions in eukaryotes through regulating downstream effectors by its product, phosphatidic acid （PA）. Protein kinases and phosphatases, such as mammalian target of rapa- mycin （mTOR）, Protein Phosphatase 1 （PP1） and Protein Phosphatase 2C （PP2C）, are PA-binding proteins that execute crucial regulatory functions in both animals and plants. PA participates in many signaling pathways by modulating the enzymatic activity and/or subcellular localization of bound proteins. In this study, we demonstrated that PLD-derived PA interacts with the scaffolding A1 subunit of Protein Phosphatase 2A （PP2A） and regulates PP2A-mediated PIN1 dephos- phorylation in Arabidopsis. Genetic and pharmacological studies showed that both PA and PP2A participate in the regu- lation of auxin distribution. In addition, both the phosphorylation status and polar localization of PIN1 protein were affected by PLD inhibitors, Exogenous PA triggered the membrane accumulation of PP2AA1 and enhanced the PP2A activity at membrane, while PLD inhibition resulted in the reduced endosomal localization and perinuclear aggregation of PP2AA1. These results demonstrate the important role of PLD-derived PA in normal PP2A-mediated PIN dephosphoryl- ation and reveal a novel mechanism, in which PA recruits PP2AA1 to the membrane system and regulates PP2A function on membrane-targeted proteins. As PA and PP2A are conserved among eukaryotes, other organisms might use similar mechanisms to mediate multiple biological processes.