Studying Protein Import into Chloroplasts Using Protoplasts

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

Pohang University of Science and Technology

Members of the epsin family of proteins (epsins) are characterized by the presence of an epsin N-terminal homology (ENTH) domain. Epsins have been implicated in various protein-trafficking pathways in animal and yeast (Saccharomyces cerevisiae) cells. Plant cells also contain multiple epsin-related proteins. In Arabidopsis (Arabidopsis thaliana), EPSIN1 is involved in vacuolar trafficking of soluble proteins. In this study, we investigated the role of Arabidopsis EpsinR2 in protein trafficking in plant cells. EpsinR2 contains a highly conserved ENTH domain but a fairly divergent C-terminal sequence. We found that the N-terminal ENTH domain specifically binds to phosphatidylinositol-3-P in vitro and has a critical role in the targeting of EpsinR2. Upon transient expression in protoplasts, hemagglutinin epitope-tagged EpsinR2 was translocated primarily to a novel cellular compartment, while a minor portion localized to the Golgi complex. Protein-binding experiments showed that EpsinR2 interacts with clathrin, AtVTI12, and the Arabidopsis homologs of adaptor protein-3 delta-adaptin and adaptor protein-2 alpha-adaptin. Localization experiments revealed that hemagglutinin epitope-tagged EpsinR2 colocalizes primarily with delta-adaptin and partially colocalizes with clathrin and AtVTI12. Based on these findings, we propose that EpsinR2 plays an important role in protein trafficking through interactions with delta-adaptin, AtVTI12, clathrin, and phosphatidylinositol-3-P.

EpsinR2 interacts with clathrin, adaptor protein-3, AtVTI12, and phosphatidylinositol-3-phosphate. Implications for EpsinR2 function in protein trafficking in plant cells.

Vacuolar sorting receptors, BP80/VSRs, play a critical role in vacuolar trafficking of soluble proteins in plant cells. However, the mechanism of action of BP80 is not well understood. Here, we investigate the action mechanism of AtVSR1, a member of BP80 proteins in Arabidopsis (Arabidopsis thaliana), in vacuolar trafficking. AtVSR1 exists as multiple forms, including a high molecular mass homomeric complex in vivo. Both the transmembrane and carboxyl-terminal cytoplasmic domains of AtVSR1 are necessary for the homomeric interaction. The carboxyl-terminal cytoplasmic domain contains specific sequence information, whereas the transmembrane domain has a structural role in the homomeric interaction. In protoplasts, an AtVSR1 mutant, C2A, that contained alanine substitution of the region involved in the homomeric interaction, was defective in trafficking to the prevacuolar compartment and localized primarily to the trans-Golgi network. In addition, overexpression of C2A, but not wild-type AtVSR1, inhibited trafficking of soluble proteins to the vacuole and caused their secretion into the medium. Furthermore, C2A:hemagglutinin in transgenic plants interfered with the homomeric interaction of endogenous AtVSR1 and inhibited vacuolar trafficking of sporamin:green fluorescent protein. These data suggest that homomeric interaction of AtVSR1 is critical for its function as a vacuolar sorting receptor.

Homomeric interaction of AtVSR1 is essential for its function as a vacuolar sorting receptor.

Although much is known about the molecular mechanisms involved in transporting soluble proteins to the central vacuole, the mechanisms governing the trafficking of membrane proteins remain largely unknown. In this study, we investigated the mechanism involved in targeting the membrane protein, AtβFructosidase 4 (AtβFruct4), to the central vacuole in protoplasts. AtβFruct4 as a green fluorescent protein (GFP) fusion protein was transported as a membrane protein during transit from the endoplasmic reticulum (ER) through the Golgi apparatus and the prevacuolar compartment (PVC). The N-terminal cytosolic domain of AtβFruct4 was sufficient for transport from the ER to the central vacuole and contained sequence motifs required for trafficking. The sequence motifs, LL and PI, were found to be critical for ER exit, while the EEE and LCPYTRL sequence motifs played roles in trafficking primarily from the trans Golgi network (TGN) to the PVC and from the PVC to the central vacuole, respectively. In addition, actin filaments and AtRabF2a, a Rab GTPase, played critical roles in vacuolar trafficking at the TGN and PVC, respectively. On the basis of these results, we propose that the vacuolar trafficking of AtβFruct4 depends on multiple sequence motifs located at the N-terminal cytoplasmic domain that function as exit and/or sorting signals in different stages during the trafficking process.

Identification of sorting motifs of AtβFruct4 for trafficking from the ER to the vacuole through the Golgi and PVC.

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In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis contains seven VSRs. Among them, VSR1, VSR3 and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, it remains controversial about the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells. Here, we provide evidence that VSR1, VSR3 and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (AALP:GFP and CPY:GFP) and PSV (phaseolin) proteins, but not a vacuolar membrane protein At[beta]Fruct4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum (ER) with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of AALP:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3 and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.

Functional identification of sorting receptors involved in trafficking of soluble lytic vacuolar proteins in vegetative cells of Arabidopsis.

Dans les cellules eucaryotes, traite de la prot&eacute;ine joue un r&ocirc;le essentiel dans la biogen&egrave;se des prot&eacute;ines qui appartiennent aux compartiments endomembranaires. Dans ce processus, une &eacute;tape importante est le tri des prot&eacute;ines organellar selon leur destination finale. Pour les prot&eacute;ines vacuolaires, vacuolaire triant des r&eacute;cepteurs (ECP) et des r&eacute;cepteurs transmembranaires homologie-anneau H2 domaine prot&eacute;ines (RMR) est consid&eacute;r&eacute;s comme responsables. Arabidopsis contient sept ECP. Parmi eux, VSR1, VSR3 et VSR4 sont impliqu&eacute;s dans le tri des prot&eacute;ines de stockage destin&eacute;s &agrave; la vacuole de stockage des prot&eacute;ines (PSV) dans les graines. Cependant, il reste controvers&eacute; sur l&#39;identit&eacute; de l&#39;ECP pour des prot&eacute;ines solubles de la vacuole lytique dans les cellules v&eacute;g&eacute;tatives. Ici, nous d&eacute;montrons que VSR1, VSR3 et VSR4 sont impliqu&eacute;s dans le tri soluble lytique vacuolaire et les prot&eacute;ines dans les cellules v&eacute;g&eacute;tatives PSV. Protoplastes de tissus foliaires de vsr1vsr3 et vsr1vsr4 mais pas vsr5vsr6, rmr1rmr2 et rmr3rmr4 double mutants, solubles lytique vacuolaire (AALP:GFP et CPY:GFP) et les prot&eacute;ines PSV (phas&eacute;oline), mais pas une prot&eacute;ine de la membrane vacuolaire &agrave; [beta] Fruct4:GFP, pr&eacute;sentait des d&eacute;fauts dans leur trafic ; ils ont accumulent dans le r&eacute;ticulum endoplasmique (ER) avec une augmentation de la s&eacute;cr&eacute;tion dans le milieu. Les d&eacute;fauts traite de protoplastes de vsr1vsr4 ont &eacute;t&eacute; secourus par VSR1 ou VSR4, mais pas VSR5 ou AtRMR1. En outre, du domaine luminal permutation entre VSR1 et VSR5, le mutant avec le domaine luminal de VSR1, mais pas celui de VSR5, des mutants ont secouru les d&eacute;fauts traite de AALP:GFP et de phas&eacute;oline vsr1vsr4 protoplastes. Selon ces r&eacute;sultats, nous proposons que les VSR1, VSR3 et VSR4, mais pas d&#39;autres ECP, participent au tri soluble lytique vacuolaire et prot&eacute;ines PSV pour leur trafic aux vacuoles dans les cellules v&eacute;g&eacute;tatives.

Identification fonctionnelle des r&eacute;cepteurs impliqu&eacute;s dans le trafic des prot&eacute;ines solubles de vacuolaires lytiques dans les cellules v&eacute;g&eacute;tatives de Arabidopsis de tri.

In eukaryotischen Zellen spielt das Protein Menschenhandel eine wesentliche Rolle bei der Biogenese der Proteine, die die Endomembrane F&auml;cher geh&ouml;ren. In diesem Prozess ist ein wichtiger Schritt beim Sortieren der organellar Proteine je nach ihren endg&uuml;ltigen Bestimmungsort. F&uuml;r welches Proteine sind welches Sortierung Rezeptoren (VSRs) und Rezeptor Homologie-Transmembran-RING H2 Dom&auml;ne Proteine (RMRs) vermutlich verantwortlich. Arabidopsis enth&auml;lt sieben VSRs. Unter ihnen sind VSR1, VSR3 und VSR4 beteiligt sortieren Speicher Proteine gezielt an das Protein Speicher Vakuole (PSV) in Samen. Es bleibt jedoch kontrovers &uuml;ber die Identit&auml;t des VSRs f&uuml;r l&ouml;sliche Proteine von der lytische Vakuole in den vegetativen Zellen. Hier wir nachweisen, dass bei der Sortierung l&ouml;sliche VSR1, VSR3 und VSR4 beteiligt sind lytische vacuolar und PSV Proteine in den vegetativen Zellen. In Urformen von Blatt Gewebe des vsr1vsr3 und vsr1vsr4 aber nicht vsr5vsr6, rmr1rmr2 und rmr3rmr4-Doppel-Mutanten, l&ouml;sliche lytischer welches (AALP:GFP und CPY:GFP) und PSV (Phaseolin) Proteine, aber keinem Vacuolar Membranprotein an [Beta] Fruct4:GFP, ausgestellten M&auml;ngel in ihren Handel; Sie angeh&auml;uft, dem Endoplasmatischen Retikulum (ER) mit eine erh&ouml;hte Sekretion in Medium. Menschenhandel M&auml;ngel vsr1vsr4 Urformen wurden von VSR1 oder VSR4 aber nicht VSR5 oder AtRMR1 gerettet. Dar&uuml;ber hinaus der luminalen Dom&auml;ne rettete Swappen Mutanten zwischen VSR1 und VSR5, den Mutanten mit der luminalen Dom&auml;ne des VSR1, aber nicht die VSR5, Menschenhandel M&auml;ngel der AALP:GFP und Phaseolin in vsr1vsr4 Urformen. Auf der Grundlage dieser Ergebnisse schlagen wir vor, VSR1, VSR3 und VSR4, aber nicht andere VSRs, Sortierung l&ouml;sliche beteiligt sind lytische vacuolar und PSV Proteine f&uuml;r ihren Handel zu den Vakuolen in den vegetativen Zellen.

Funktionale Identifikation von Rezeptoren beteiligt, Menschenhandel l&ouml;sliche lytischer welches Proteine in den vegetativen Zellen der Arabidopsis sortieren.

Nelle cellule eucariotiche, traffico della proteina svolge un ruolo essenziale nella biogenesi delle proteine che appartengono ai vani di endomembrane. In questo processo, un passo importante &egrave; l&#39;ordinamento delle proteine organellare a seconda della loro destinazione finale. Per proteine vacuolare, vacuolar ordinamento recettori (VSRs) e del recettore transmembrana omologia-anello H2 dominio proteine (RMRs) si pensa siano responsabili. Arabidopsis contiene sette VSRs. Tra di loro, VSR1, VSR3 e VSR4 sono coinvolti nelle proteine di immagazzinaggio mirate per il vacuolo di stoccaggio della proteina (PSV) in semi di ordinamento. Tuttavia, rimane controversa circa l&#39;identit&agrave; del VSRs per le proteine solubili del vacuolo litico in cellule vegetative. Qui, ci forniscono prove che VSR1, VSR3 e VSR4 sono coinvolti nell&#39;ordinamento solubile litico vacuolar e PSV proteine in cellule vegetative. In protoplasti dai tessuti di foglie di vsr1vsr3 e vsr1vsr4 ma non vsr5vsr6, rmr1rmr2 e rmr3rmr4 doppio mutanti, solubile litico vacuolar (AALP:GFP e CPY:GFP) e PSV (faseolina) proteine, ma non una proteina di membrana vacuolar presso Fruct4:GFP [beta], difetti esposti nei loro traffici; hanno accumulato al reticolo endoplasmatico (ER) con un&#39;aumentata secrezione nel mezzo. I difetti traffici in protoplasti di vsr1vsr4 furono salvati da VSR1 o VSR4 ma non VSR5 o AtRMR1. Inoltre, del dominio Luminale swapping mutanti tra VSR1 e VSR5, il mutante con il dominio Luminale di VSR1, ma non quella di VSR5, salvo i traffici difetti di faseolina in protoplasti di vsr1vsr4 e di AALP:GFP. Basato su questi risultati, proponiamo che VSR1, VSR3 e VSR4, ma non altri VSRs, sono coinvolti nell&#39;ordinamento solubile litico vacuolar e PSV proteine per il loro traffico di vacuoli in cellule vegetative.

Identificazione funzionale dell&#39;ordinamento recettori coinvolti nel traffico di proteine solubili litiche vacuolare in cellule vegetative di Arabidopsis.

&#x771F;&#x6838;&#x751F;&#x7269;&#x306E;&#x7D30;&#x80DE;&#x5185;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x8F38;&#x9001;&#x5F13;&#x90E8;&#x30B3;&#x30F3;&#x30D1;&#x30FC;&#x30C8;&#x30E1;&#x30F3;&#x30C8;&#x306B;&#x5C5E;&#x3059;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x751F;&#x5408;&#x6210;&#x306B;&#x304A;&#x3051;&#x308B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3067;&#x306F;&#x3001;&#x91CD;&#x8981;&#x306A;&#x30B9;&#x30C6;&#x30C3;&#x30D7;&#x306F;&#x3001;&#x6700;&#x7D42;&#x7684;&#x306A;&#x9001;&#x4FE1;&#x5148;&#x306B;&#x5FDC;&#x3058;&#x3066;&#x30AA;&#x30EB;&#x30AC;&#x30CD;&#x30E9;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x3067;&#x3059;&#x3002;&#x6DB2;&#x80DE;&#x306E;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x305F;&#x3081;&#x6DB2;&#x80DE;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x53D7;&#x5BB9;&#x4F53; (VSRs) &#x304A;&#x3088;&#x3073;&#x53D7;&#x5BB9;&#x4F53;&#x76F8;&#x540C;&#x6027;&#x819C;&#x8CAB;&#x901A;&#x30EA;&#x30F3;&#x30B0; H2 &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x86CB;&#x767D;&#x8CEA; (RMRs) &#x8CAC;&#x4EFB;&#x304C;&#x3042;&#x308B;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x307E;&#x3059;&#x3002;&#x30B7;&#x30ED;&#x30A4;&#x30CC;&#x30CA;&#x30BA;&#x30CA;&#x306B;&#x306F;&#x3067;&#x306F; 7 &#x3064;&#x306E; VSRs &#x304C;&#x542B;&#x307E;&#x308C;&#x307E;&#x3059;&#x3002;&#x8CAF;&#x8535;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x7A2E;&#x5B50;&#x306B;&#x304A;&#x3051;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30B9;&#x30C8;&#x30EC;&#x30FC;&#x30B8;&#x80DE; (PSV) &#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x306E;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x3067;&#x305D;&#x308C;&#x3089;&#x306E;&#x9593;&#x306B;&#x3001;VSR1&#x3001;VSR3&#x3001;VSR4 &#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x6804;&#x990A;&#x7D30;&#x80DE;&#x6EB6;&#x89E3;&#x6DB2;&#x80DE;&#x306E;&#x53EF;&#x6EB6;&#x6027;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x305F;&#x3081;&#x306E; VSRs &#x306E;&#x30A2;&#x30A4;&#x30C7;&#x30F3;&#x30C6;&#x30A3;&#x30C6;&#x30A3;&#x306B;&#x3064;&#x3044;&#x3066;&#x8B70;&#x8AD6;&#x306E;&#x4F59;&#x5730;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F; VSR1&#x3001;VSR3 &#x304A;&#x3088;&#x3073; VSR4 &#x53EF;&#x6EB6;&#x6027;&#x306E;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x306E;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x6EB6;&#x89E3;&#x6DB2;&#x80DE;&#x3068; PSV &#x86CB;&#x767D;&#x8CEA;&#x6804;&#x990A;&#x7D30;&#x80DE;&#x306E;&#x3002;Vsr1vsr3 &#x3068; vsr1vsr4 &#x304C;&#x3044;&#x306A;&#x3044; vsr5vsr6 &#x306E;&#x8449;&#x7D44;&#x7E54;&#x304B;&#x3089;&#x306E;&#x30D7;&#x30ED;&#x30C8;&#x30D7; &#x30E9;&#x30B9;&#x30C8;&#x3067; rmr1rmr2 &#x3068; rmr3rmr4 &#x306E;&#x4E8C;&#x91CD;&#x5909;&#x7570;&#x4F53;&#x3001;&#x6C34;&#x6EB6;&#x6027;&#x6EB6;&#x83CC;&#x6DB2;&#x80DE; (AALP:GFP &#x304A;&#x3088;&#x3073; CPY:GFP) &#x3068; PSV (phaseolin) &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x304C;&#x306A;&#x3044;&#x6DB2;&#x80DE;&#x819C;&#x86CB;&#x767D;&#x8CEA; [&#x30D9;&#x30FC;&#x30BF;&#x7248;] Fruct4:GFP &#x3067;&#x5C55;&#x793A;&#x306E;&#x6B20;&#x9665;&#x3001;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;;&#x5F7C;&#x3089;&#x3092;&#x5A92;&#x4F53;&#x306B;&#x5897;&#x52A0;&#x5206;&#x6CCC;&#x5C0F;&#x80DE;&#x4F53; &#xFF08;ER&#xFF09; &#x3092;&#x84C4;&#x7A4D;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;Vsr1vsr4 &#x30D7;&#x30ED;&#x30C8;&#x30D7; &#x30E9;&#x30B9;&#x30C8;&#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E;&#x6B20;&#x9665;&#x306F;&#x3001;VSR1 &#x307E;&#x305F;&#x306F; VSR4 &#x304C;&#x306A;&#x3044; VSR5 &#x307E;&#x305F;&#x306F; AtRMR1 &#x306B;&#x3088;&#x3063;&#x3066;&#x6551;&#x51FA;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x5185;&#x8154;&#x306E;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x5909;&#x7570;&#x4F53; VSR1 &#x3068; VSR5&#x3001;VSR1 &#x306E;&#x5185;&#x8154;&#x306E;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x304C;&#x306A;&#x3044;&#x3053;&#x3068; VSR5 &#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x4F53;&#x306E;&#x9593;&#x306E;&#x30B9;&#x30EF;&#x30C3;&#x30D7; AALP:GFP &#x3068; phaseolin vsr1vsr4 &#x30D7;&#x30ED;&#x30C8;&#x30D7; &#x30E9;&#x30B9;&#x30C8;&#x3067;&#x306E;&#x8F38;&#x9001;&#x6B20;&#x9665;&#x3092;&#x6551;&#x52A9;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x3001;&#x6211;&#x3005; VSR1&#x3001;VSR3&#x3001;VSR4 &#x304C;&#x306A;&#x3044;&#x4ED6;&#x306E; VSRs &#x304C;&#x53EF;&#x6EB6;&#x6027;&#x306E;&#x4E26;&#x3079;&#x66FF;&#x3048;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x63D0;&#x6848;&#x3059;&#x308B;&#x6EB6;&#x89E3;&#x6DB2;&#x80DE;&#x3068;&#x305D;&#x306E;&#x6804;&#x990A;&#x7D30;&#x80DE;&#x306E;&#x7A7A;&#x80DE;&#x306B;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E; PSV &#x86CB;&#x767D;&#x8CEA;&#x3002;

&#x53D7;&#x5BB9;&#x4F53;&#x53EF;&#x6EB6;&#x6027;&#x306E;&#x6EB6;&#x89E3;&#x6DB2;&#x80DE;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30B7;&#x30ED;&#x30A4;&#x30CC;&#x30CA;&#x30BA;&#x30CA;&#x306E;&#x6804;&#x990A;&#x7D30;&#x80DE;&#x3067;&#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306B;&#x95A2;&#x4E0E;&#x30BD;&#x30FC;&#x30C8;&#x306E;&#x6A5F;&#x80FD;&#x7684;&#x540C;&#x5B9A;

&#xC9C4; &#xD575; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xB2E8;&#xBC31;&#xC9C8; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4; endomembrane &#xAD6C;&#xD68D;&#xC5D0; &#xC18D;&#xD558;&#xB294; &#xB2E8;&#xBC31;&#xC9C8; &#xC18D;&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. &#xC774; &#xACFC;&#xC815;&#xC5D0;&#xC11C; &#xC911;&#xC694; &#xD55C; &#xB2E8;&#xACC4;&#xB294; &#xADF8;&#xB4E4;&#xC758; &#xCD5C;&#xC885; &#xBAA9;&#xC801;&#xC9C0;&#xC5D0; &#xB530;&#xB77C; organellar &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC815;&#xB82C;. Vacuolar &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xB300; &#xD55C; &#xC815;&#xB82C; (VSRs) &#xC218;&#xC6A9; &#xCCB4;&#xC640; &#xC218;&#xC6A9; &#xCCB4; homology &#xB9C9; &#xD6A1;&#xB2E8; &#xB9C1; h 2 &#xB3C4;&#xBA54;&#xC778; &#xB2E8;&#xBC31;&#xC9C8; vacuolar (RMRs) &#xAC83;&#xC73C;&#xB85C; &#xC0DD;&#xAC01; &#xB429;&#xB2C8;&#xB2E4; &#xCC45;&#xC784;. Arabidopsis 7 VSRs &#xD3EC;&#xD568; &#xB418;&#xC5B4; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8; &#xC911; VSR1, VSR3 &#xBC0F; vsr4&#xB294; &#xC815;&#xB82C; &#xD558;&#xB294; &#xC528;&#xC557;&#xC5D0; &#xB2E8;&#xBC31;&#xC9C8; &#xC800;&#xC7A5; &#xACF5;&#xD3EC; (PSV)&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xC800;&#xC7A5; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xCC38;&#xC5EC;. &#xADF8;&#xB7EC;&#xB098; &#xC6A9;&#xADE0;&#xC131; &#xACF5;&#xD3EC; &#xC2DD;&#xBB3C; &#xC138;&#xD3EC;&#xC5D0;&#xC11C;&#xC758; &#xC218;&#xC6A9; &#xC131; &#xB2E8;&#xBC31;&#xC9C8; VSRs id&#xC5D0; &#xB300; &#xD55C; &#xB17C;&#xB780;&#xC774; &#xC788;&#xB2E4;. VSR1, VSR3 &#xBC0F; VSR4 &#xC218;&#xC6A9; &#xC131; &#xC815;&#xB82C;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xC5EC;&#xAE30;, &#xC6A9;&#xADE0;&#xC131; vacuolar&#xACFC; &#xC2DD;&#xBB3C; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; PSV&#xB294; &#xB2E8;&#xBC31;&#xC9C8;. Vsr1vsr3 &#xBC0F; vsr1vsr4 &#xD558;&#xC9C0; vsr5vsr6&#xC758; &#xC78E; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; protoplasts, rmr1rmr2 &#xBC0F; rmr3rmr4 &#xB450; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;, &#xC218;&#xC6A9; &#xC131; &#xC6A9;&#xADE0;&#xC131; vacuolar (AALP:GFP &#xBC0F; CPY:GFP) &#xBC0F; PSV (phaseolin) &#xB2E8;&#xBC31;&#xC9C8;, &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; [&#xBCA0;&#xD0C0;] fruct4:gfp&#xC5D0;&#xC11C; vacuolar &#xB9C9; &#xB2E8;&#xBC31;&#xC9C8; &#xC804;&#xC2DC; &#xACB0;&#xD568;&#xC758; &#xBC00;&#xB9E4;; &#xADF8;&#xB4E4;&#xC740; &#xB9E4;&#xCCB4;&#xB85C; &#xC99D;&#xAC00; &#xBD84; &#xBE44;&#xC640; endoplasmic &#xADF8;&#xBB3C; (&#xC751;&#xAE09;&#xC2E4;)&#xC744; &#xCD95;&#xC801;. &#xBC00;&#xB9E4; &#xACB0;&#xD568; vsr1vsr4 protoplasts&#xC5D0; VSR5 &#xB610;&#xB294; AtRMR1 &#xD558;&#xC9C0; VSR1 &#xB610;&#xB294; vsr4&#xC5D0; &#xC758;&#xD574; &#xAD6C;&#xCD9C; &#xB418;&#xC5C8;&#xB2E4;. &#xB610;&#xD55C;, luminal &#xB3C4;&#xBA54;&#xC778;&#xC758; AALP:GFP &#xBC0F; vsr1vsr4 protoplasts&#xC5D0;&#xC11C; phaseolin&#xC758; &#xBC00;&#xB9E4; &#xACB0;&#xD568; &#xAD6C;&#xC870; vsr1&#xC640; VSR5, VSR1, luminal &#xB3C4;&#xBA54;&#xC778; &#xD558;&#xC9C0;&#xB9CC; &#xADF8; VSR5, &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xAD50;&#xD658;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xBC14;&#xD0D5;&#xC73C;&#xB85C;, &#xC6B0;&#xB9AC; VSR1, VSR3 &#xBC0F; VSR4, &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; &#xB2E4;&#xB978; VSRs &#xB179;&#xB294; &#xC815;&#xB82C;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xC81C;&#xC548; &#xC6A9;&#xADE0;&#xC131; vacuolar &#xBC0F; &#xADF8;&#xB4E4;&#xC758; &#xC2DD;&#xBB3C; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; vacuoles&#xC744; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xB300; &#xD55C; PSV &#xB2E8;&#xBC31;&#xC9C8;.

&#xC815;&#xB82C; Arabidopsis&#xC758; &#xC2DD;&#xBB3C; &#xC138;&#xD3EC;&#xC5D0; &#xC788;&#xB294; &#xC218;&#xC6A9; &#xC131; &#xC6A9;&#xADE0;&#xC131; vacuolar &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xAE30;&#xB2A5; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;.

En las c&eacute;lulas eucariotas, trata de prote&iacute;na juega un papel esencial en la biog&eacute;nesis de prote&iacute;nas que pertenecen a los compartimentos endomembranoso. En este proceso, un paso importante es la clasificaci&oacute;n de las prote&iacute;nas organellar dependiendo de su destino final. Prote&iacute;nas vacuolar, vacuolar clasificaci&oacute;n de receptores (VSRs) y prote&iacute;nas de dominio receptor transmembrana homolog&iacute;a-anillo H2 (RMRs) se cree que son responsables. Arabidopsis contiene siete VSRs. Entre ellos, VSR1, VSR3 y VSR4 participan en la clasificaci&oacute;n de las prote&iacute;nas de almacenamiento de informaci&oacute;n dirigidas a la vacuola de almacenamiento de la prote&iacute;na (PSV) en semillas. Sin embargo, sigue siendo pol&eacute;mica sobre la identidad de VSRs por prote&iacute;nas solubles de la vacuola l&iacute;tica en c&eacute;lulas vegetativas. Aqu&iacute; proporcionamos evidencia que VSR1, VSR3 y VSR4 participan en clasificaci&oacute;n soluble l&iacute;ticas vacuolar y PSV prote&iacute;nas en c&eacute;lulas vegetativas. En protoplastos de tejidos de la hoja de vsr1vsr3 y vsr1vsr4 pero no vsr5vsr6, rmr1rmr2 y rmr3rmr4 dobles mutantes, solubles l&iacute;ticas vacuolar (AALP:GFP y CPY:GFP) y prote&iacute;nas PSV (faseolina), pero no una prote&iacute;na de membrana vacuolares en Fruct4:GFP [beta], defectos expuestos en su tr&aacute;fico; acumula hasta el ret&iacute;culo endopl&aacute;smico (ER) con una secreci&oacute;n aumentada en medio. Los defectos de tr&aacute;fico en protoplastos de vsr1vsr4 fueron rescatados por VSR1 o VSR4 pero no VSR5 o AtRMR1. Adem&aacute;s, el dominio luminal intercambio mutantes entre VSR1 y VSR5, el mutante con el dominio luminal de VSR1, pero no la de VSR5, rescat&oacute; los defectos trata de AALP:GFP y faseolina en protoplastos de vsr1vsr4. Basado en estos resultados, proponemos que VSR1, VSR3 y VSR4, pero no otros VSRs, participan en la clasificaci&oacute;n soluble l&iacute;ticas vacuolar y PSV prote&iacute;nas para su tr&aacute;fico a las vacuolas en c&eacute;lulas vegetativas.

Identificaci&oacute;n funcional de clasificaci&oacute;n de receptores implicados en tr&aacute;fico de prote&iacute;nas solubles de vacuolares l&iacute;ticas en c&eacute;lulas vegetativas de Arabidopsis.

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.

Trafficking of vacuolar proteins: the crucial role of Arabidopsis vacuolar protein sorting 29 in recycling vacuolar sorting receptor.

Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2-dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.

Adaptor protein complex 2-mediated endocytosis is crucial for male reproductive organ development in Arabidopsis.

The nucleotide sequence around the translational initiation site is an important cis-acting element for post-transcriptional regulation. However, it has not been fully understood how the sequence context at the 5'-untranslated region (5'-UTR) affects the translational efficiency of individual mRNAs. In this study, we provide evidence that the 5'-UTRs of Arabidopsis genes showing a great difference in the nucleotide sequence vary greatly in translational efficiency with more than a 200-fold difference. Of the four types of nucleotides, the A residue was the most favourable nucleotide from positions -1 to -21 of the 5'-UTRs in Arabidopsis genes. In particular, the A residue in the 5'-UTR from positions -1 to -5 was required for a high-level translational efficiency. In contrast, the T residue in the 5'-UTR from positions -1 to -5 was the least favourable nucleotide in translational efficiency. Furthermore, the effect of the sequence context in the -1 to -21 region of the 5'-UTR was conserved in different plant species. Based on these observations, we propose that the sequence context immediately upstream of the AUG initiation codon plays a crucial role in determining the translational efficiency of plant genes. 

The immediate upstream region of the 5'-UTR from the AUG start codon has a pronounced effect on the translational efficiency in Arabidopsis thaliana.

Vacuoles are one of the most prominent organelles in plant cells, and they play various important roles, such as degradation of waste materials, storage of ions and metabolites, and maintaining turgor. During the past two decades, numerous advances have been made in understanding how proteins are specifically delivered to the vacuole. One of the most crucial steps in this process is specific sorting of soluble vacuolar proteins. Vacuolar sorting receptors (VSRs), which are type I membrane proteins, are involved in the sorting and packaging of soluble vacuolar proteins into transport vesicles with the help of various accessory proteins. To date, large amounts of data have led to the development of two different models describing VSR-mediated vacuolar trafficking that are radically different in multiple ways, particularly regarding the location of cargo binding to, and release from, the VSR and the types of carriers utilized. In this review, we summarize current literature aimed at elucidating VSR-mediated vacuolar trafficking and compare the two models with respect to the sorting signals of vacuolar proteins, as well as the molecular machinery involved in VSR-mediated vacuolar trafficking and its action mechanisms. 

Vacuolar Sorting Receptor-Mediated Trafficking of Soluble Vacuolar Proteins in Plant Cells.

Plants represent promising systems for producing various recombinant proteins. One key area of focus for improving this technology is developing methods for producing recombinant proteins at high levels. Many methods have been developed to increase the transcript levels of recombinant genes. However, methods for increasing protein production involving steps downstream of transcription, including translation, have not been fully explored. Here, we investigated the effects of N-glycosylation on protein production and provide evidence that N-glycosylation greatly increases the expression levels of ER-targeted recombinant proteins. Fusion of the extracellular domain (M domain) of protein tyrosine phosphatase receptor type C (CD45), which contains four putative N-glycosylation sites to a model protein, leptin at the C-terminus, increased recombinant protein levels by 6.1 fold. This increase was specific to ER-targeted proteins and was dependent on N-glycosylation. Moreover, expression levels of leptin, leukemia inhibitory factor and GFP were also greatly increased by fusion of M domain at either the N or C-terminus. Furthermore, the increase in protein levels resulted from enhanced translation, but not transcription. Based on these results, we propose that fusing a small domain containing N-glycosylation sites to target proteins is a powerful technique for increasing the expression levels of recombinant proteins in plants.

Hyangju Kang

Division of Integrative Biosciences and Biotechnology

Pohang University of Science and Technology

Studying Protein Import into Chloroplasts Using Protoplasts

Hyangju Kang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Integrative Biosciences and Biotechnology

Pohang University of Science and Technology

Studying Protein Import into Chloroplasts Using Protoplasts

Division of Integrative Biosciences and Biotechnology