Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells

We generated fusions between three Arabidopsis (Arabidopsis thaliana) tonoplast intrinsic proteins (TIPs; alpha-, gamma-, and delta-TIP) and yellow fluorescent protein (YFP). We also produced soluble reporters consisting of the monomeric red fluorescent protein (RFP) and either the C-terminal vacuolar sorting signal of phaseolin or the sequence-specific sorting signal of proricin. In transgenic Arabidopsis leaves, mature roots, and root tips, all TIP fusions localized to the tonoplast of the central vacuole and both of the lumenal RFP reporters were found within TIP-delimited vacuoles. In embryos from developing, mature, and germinating seeds, all three TIPs localized to the tonoplast of protein storage vacuoles. To determine the temporal TIP expression patterns and to rule out mistargeting due to overexpression, we generated plants expressing YFP fused to the complete genomic sequences of the three TIP isoforms. In transgenic Arabidopsis, gamma-TIP expression was limited to vegetative tissues, but specifically excluded from root tips, whereas alpha-TIP was exclusively expressed during seed maturation. delta-TIP was expressed in vegetative tissues, but not root tips, at a later stage than gamma-TIP. Our findings indicate that, in the Arabidopsis tissues analyzed, two different vacuolar sorting signals target soluble proteins to a single vacuolar location. Moreover, TIP isoform distribution is tissue and development specific, rather than organelle specific.     

Protein sorting to the vacuolar membrane.

The vacuolar membrane (tonoplast) of plant cells contains a polytopic integral membrane protein with six membrane-spanning domains and cytoplasmically oriented amino-terminal and carboxy-terminal domains. This protein, tonoplast intrinsic protein (TIP), is a member of the membrane intrinsic protein (MIP) family of proteins, a family of channel proteins found in a variety of organisms. In bean seeds, alpha-TIP is synthesized on the rough endoplasmic reticulum and its transport to the tonoplast is mediated by the secretory system. In this study, we report that a polypeptide segment that includes the sixth membrane domain and the cytoplasmic tail of 18 amino acids of alpha-TIP is sufficient to target the reporter protein phosphinotricine acetyltransferase to the tonoplast of stably transformed tobacco cells. To determine if the carboxy-terminal cytoplasmic tail of alpha-TIP contains important tonoplast targeting information, a deletion construct lacking the 15 carboxy-terminal amino acids was introduced for transient expression in tobacco cells; we found that the slightly truncated protein still accumulated in the tonoplast. From these results, we concluded that a transmembrane domain of a tonoplast protein probably contains sufficient information for transport to the tonoplast. Whether such transport occurs by bulk flow or involves specific cellular machinery remains to be determined.     

Lack of a vacuolar sorting receptor leads to non-specific missorting of soluble vacuolar proteins in Arabidopsis seeds

The plant vacuolar sorting receptor (VSR) binds proteins carrying vacuolar sorting signals (VSS) of the 'sequence-specific' type (ssVSS) but not the C-terminal, hydrophobic sorting signals (ctVSS). Seeds of Arabidopsis mutants lacking the major VSR isoform, AtVSR1, secrete a proportion of the proteins destined to storage vacuoles. The sorting signals for these proteins are not well defined, but they do not seem to be of the ssVSS type. Here, we tested whether absence of VSR1 in seeds leads to secretion of reporter proteins carrying ssVSS but not ctVSS. Our results show that reporters carrying either ssVSS or ctVSS are equally secreted in the absence of VSR1. We discuss our findings in relation to the current model for vacuolar sorting.     

The first membrane spanning region of the lamin B receptor is sufficient for sorting to the inner nuclear membrane

The lamin B receptor (LBR) is a polytopic integral membrane protein localized exclusively in the inner nuclear membrane domain of the nuclear envelope. Its cDNA deduced primary structure consists of a highly charged amino-terminal domain of 205 residues that faces the nucleoplasm followed by a hydrophobic domain with eight potential transmembrane segments. To identify determinants that sort LBR from its site of integration (RER and outer nuclear membrane) to the inner nuclear membrane, we prepared full-length, truncated, and chimeric cDNA constructs of chick LBR, transfected these into mammalian cells and detected the expressed protein by immunofluorescence microscopy using appropriate antibodies. Surprisingly, we found that the determinants for sorting of LBR to the inner nuclear membrane reside in a region comprising its first transmembrane sequence plus flanking residues on either side. The other transmembrane regions as well as the nucleoplasmic domain are not required for sorting. We propose that the first transmembrane segment of LBR interacts specifically with another transmembrane segment and consider several mechanisms by which such specific interaction could result in sorting to the inner nuclear membrane.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.     

N‐linked glycosylation of AtVSR1 is important for vacuolar protein sorting in Arabidopsis

Vacuolar sorting receptors (VSRs) in Arabidopsis mediate the sorting of soluble proteins to vacuoles in the secretory pathway. The VSRs are post‐translationally modified by the attachment of N‐glycans, but the functional significance of such a modification remains unknown. Here we have studied the role(s) of glycosylation in the stability, trafficking and vacuolar protein transport of AtVSR1 in Arabidopsis protoplasts. AtVSR1 harbors three complex‐type N‐glycans, which are located in the N‐terminal ‘PA domain’, the central region and the C‐terminal epidermal growth factor repeat domain, respectively. We have demonstrated that: (i) the N‐glycans do not affect the targeting of AtVSR1 to pre‐vacuolar compartments (PVCs) and its vacuolar degradation; and (ii) N‐glycosylation alters the binding affinity of AtVSR1 to cargo proteins and affects the transport of cargo into the vacuole. Hence, N‐glycosylation of AtVSR1 plays a critical role in its function as a VSR in plants.     

The N-terminal ATPase AT-hook-containing region of the Arabidopsis chromatin-remodeling protein SPLAYED is sufficient for biological activity

The SNF2-like chromatin-remodeling ATPase SPLAYED (SYD) was identified as a co-activator of floral homeotic gene expression in Arabidopsis. SYD is also required for meristem maintenance and regulates flowering under a non-inductive photoperiod. SNF2 ATPases are structurally and functionally conserved from yeast to humans. In addition to the conserved protein features, SYD has a large unique C-terminal domain. We show here that SYD is present as two forms in the nucleus, full-length and truncated, with the latter apparently lacking the C-terminal domain. The ratio of the two forms of endogenous SYD differs in juvenile and in adult tissues. Furthermore, an SYD variant lacking the C-terminal domain (SYDDeltaC) rescues the syd null mutant, indicating that the N-terminal ATPase AT-hook-containing region of SYD is sufficient for biological activity. Plants expressing SYDDeltaC show molecular and morphological phenotypes opposite to those of the null mutant, suggesting that the construct results in increased activity. This increased activity is at least in part due to elevated SYD protein levels in these lines. We propose that the C-terminal domain may control SYD accumulation and/or specific activity in the context of the full-length protein. The presence of the C-terminal domain in rice SYD suggests that its role is probably conserved in the two classes of flowering plants.     

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana.

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana have been studied at the single-channel level using the patch-clamp technique. The Ca2+ channel in the plasma membrane opened for extracellular Ca2+ influx. The Ca2+ channel in the vacuolar membrane opened for cytoplasmic Ca2+ influx.     

Relationship of the membrane ATPase from Halobacterium saccharovorum to vacuolar ATPases.

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan inhibited the halobacterial ATPase also in a nucleotide-protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuolar and the F-type ATPases.     

The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope

The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.     

Structural determinants for rCNT2 sorting to the plasma membrane of polarized and non-polarized cells

rCNT2 (rat concentrative nucleoside transporter 2) (Slc28a2) is a purine-preferring concentrative nucleoside transporter. It is expressed in both non-polarized and polarized cells, where it is localized in the brush border membrane. Since no information about the domains implicated in the plasma membrane sorting of rCNT2 is available, the present study aimed to identify structural and functional requirements for rCNT2 trafficking. The comprehensive topological mapping of the intracellular N-terminal tail revealed two main features: (i) a glutamate-enriched region (NPGLELME) between residues 21 and 28 that seems to be implicated in the stabilization of rCNT2 in the cell surface, since mutagenesis of these conserved glutamates resulted in enhanced endocytosis; and (ii) mutation of a potential protein kinase CK2 domain that led to a loss of brush border-specific sorting. Although the shortest proteins assayed (rCNT2-74AA, -48AA and -37AA) accumulated intracellularly and lost their brush border membrane preference, they were still functional. A deeper analysis of CK2 implication in CNT2 trafficking, using a CK2-specific inhibitor [DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole)] and other complementary mutations mimicking the negative charge provided by phosphorylation (S46D and S46E), demonstrated an effect of this kinase on rCNT2 activity. In summary, the N-terminal tail of rCNT2 contains dual sorting signals. An acidic region is responsible for its proper stabilization at the plasma membrane, whereas the putative CK2 domain (Ser(46)) is implicated in the apical sorting of the transporter.     

Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase.

Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.     

Structure and function of the yeast vacuolar membrane proton ATPase

Our current work on a vacuolar membrane proton ATPase in the yeastSaccharomyces cerevisiae has revealed that it is a third type of H⁺-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F₀F₁-type ATP synthase and the plasma membrane E₁E₂-type H⁺-ATPase. The vacuolar membrane H⁺-ATPase is composed of three major subunits, subunita (M _r =67 kDa),b (57kDa), andc (20 kDa). Subunita is the catalytic site and subunitc functions as a channel for proton translocation in the enzyme complex. The function of subunitb has not yet been identified. The functional molecular masses of the H⁺-ATPase under two kinetic conditions have been determined to be 0.9–1.1×10⁵ daltons for single-cycle hydrolysis of ATP and 4.1–5.3×10⁵ daltons for multicycle hydrolysis of ATP, respectively.N,N-Dicyclohexylcarbodiimide does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazolesensitive catalytic site. Cloning of structural genes for the three subunits of the H⁺-ATPase (VMA1, VMA2, andVMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.Abbreviations CCCP carbonyl cyanidem-chlorophenyl hydrazone - DCCD N,N-dicyclohexylcarbondiimide - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-stilbene disulfonic acid - NBD-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Pi inorganic phosphate - SDS sodium dodecylsulfate - SF6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile - SITS 4-acetamide-4-isothiocyanatostilbene-2,2-disulfonic acid - ZW3-14 N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate     

Evidence for a sorting endosome in Arabidopsis root cells

In eukaryotic cells, the endocytic and secretory pathways are key players in several physiological processes. These pathways are largely inter-connected in animal and yeast cells through organelles named sorting endosomes. Sorting endosomes are multi-vesicular compartments that redirect proteins towards various destinations, such as the lysosomes or vacuoles for degradation, the trans-Golgi network for retrograde transport and the plasma membrane for recycling. In contrast, cross-talk between the endocytic and secretory pathways has not been clearly established in plants, especially in terms of cargo protein trafficking. Here we show by co-localization analyses that endosomes labelled with the AtSORTING NEXIN1 (AtSNX1) protein overlap with the pre-vacuolar compartment in Arabidopsis root cells. In addition, alteration of the routing functions of AtSNX1 endosomes by drug treatments leads to mis-routing of endocytic and secretory cargo proteins. Based on these results, we propose that the AtSNX1 endosomal compartment represents a sorting endosome in root cells, and that this specialized organelle is conserved throughout eukaryotes.     

Ubiquitin-mediated targeting of a mutant plasma membrane ATPase, Pma1-7, to the endosomal/vacuolar system in yeast

Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37 degrees C and is delivered instead to the endosomal/vacuolar pathway for degradation. We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism. By contrast with wild-type Pma1, Pma1-7 is ubiquitinated. Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1. Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry. In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable. Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts. In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface. On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface. We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.     

The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana

Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH(2)-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.     

An expanded and flexible form of the vacuolar ATPase membrane sector

The vacuolar ATPase integral membrane c-ring from Nephrops norvegicus occurs in paired complexes in a double membrane. Using cryo-electron microscopy and single particle image processing of 2D crystals, we have obtained a projection structure of the c-ring of N. norvegicus. The c-ring was found to be very flexible, most likely as a result of an expanded conformation of the c subunits. This structure may support a role for the vacuolar ATPase c-rings in membrane fusion.