Vacuolar Targeting Research Articles

Endocytosis-Mediated Vacuolar Accumulation of the Human ApoE Apolipoprotein-Derived ApoEdpL-W Antimicrobial Peptide Contributes to Its Antifungal Activity in Candida albicans

The 18-amino-acid cationic, tryptophan-rich ApoEdpL-W peptide derived from human ApoE apolipoprotein was shown to have antifungal activity against pathogenic yeasts of the Candida genus (except C. glabrata). ApoEdpL-W was active against planktonic cells and early-stage biofilms but less active against mature biofilms, possibly because of its affinity for extracellular matrix beta-glucans. Moreover, ApoEdpL-W absorbed to medically relevant materials partially prevented the formation of biofilms on these materials. The exposure of C. albicans cells to sublethal doses of ApoEdpL-W triggered a transcriptional response reminiscent of that associated with the inactivation of the MYO5 gene required for endocytosis as well as the upregulation of amino acid transporter genes. A fluorescent derivative of ApoEdpL-W accumulated at the cytoplasmic membrane and subsequently was translocated to the vacuole. Strikingly, the inactivation of MYO5 or addition of latrunculin, an inhibitor of endocytosis, prevented the vacuolar accumulation of fluorescein-labeled ApoEdpL-W and reduced the antifungal activity of ApoEdpL-W. This, together with the insensitivity of ApoEdpL-W to alterations in membrane fluidity and high salt, suggested that the ApoEdpL-W mode of action was dependent upon vacuolar targeting and differed significantly from that of other antifungal peptides, such as Histatin-5 and Magainin 2.

Structure and Mechanism of the Saposin-like Domain of a Plant Aspartic Protease

Many plant aspartic proteases contain an additional sequence of ~100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plant-specific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å, revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus.

High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene

Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10 4 fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30 mg g −1 of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.

Aspartyl Aminopeptidase Is Imported from the Cytoplasm to the Vacuole by Selective Autophagy in Saccharomyces cerevisiae

Macroautophagy is a catabolic process by which cytosolic components are sequestered by double membrane vesicles called autophagosomes and sorted to the lysosomes/vacuoles to be degraded. Saccharomyces cerevisiae has adapted this mechanism for constitutive transport of the specific vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1); this process is called the cytoplasm to vacuole targeting (Cvt) pathway. The precursor form of Ape1 self-assembles into an aggregate-like structure in the cytosol that is then recognized by Atg19 in a propeptide-dependent manner. The interaction between Atg19 and autophagosome-forming machineries allows selective packaging of the Ape1-Atg19 complex by the autophagosome-like Cvt vesicle. Ams1 also forms oligomers and utilizes the Ape1 transport system by interacting with Atg19. Although the mechanism of selective transport of the Cvt cargoes has been well studied, it is unclear whether proteins other than Ape1 and Ams1 are transported via the Cvt pathway. We describe here that aspartyl aminopeptidase (Yhr113w/Ape4) is the third Cvt cargo, which is similar in primary structure and subunit organization to Ape1. Ape4 has no propeptide, and it does not self-assemble into aggregates. However, it binds to Atg19 in a site distinct from the Ape1- and Ams1-binding sites, allowing it to "piggyback" on the Ape1 transport system. In growing conditions, a small portion of Ape4 localizes in the vacuole, but its vacuolar transport is accelerated by nutrient starvation, and it stably resides in the vacuole lumen. We propose that the cytosolic Ape4 is redistributed to the vacuole when yeast cells need more active vacuolar degradation.

A New Autophagy-related Checkpoint in the Degradation of an ERAD-M Target

The endoplasmic reticulum (ER) harbors elaborate quality control mechanisms to ensure proper folding and post-translational modifications of polypeptides targeted to this organelle. Once an aberrant protein is detected, it is dislocated from the ER and routed to the proteasome for destruction. Autophagy has been recently implicated in the elevation of the ER stress response; however, the involvement of this pathway in selective removal of ER-associated degradation (ERAD) substrates has not been demonstrated. In the present study, we show that an ER membrane lesion, associated with the accumulation of the yeast ERAD-M substrate 6Myc-Hmg2p elicits the recruitment of Atg8 and elements of the cytosol to vacuole targeting (CVT) to the membrane, leading to attenuation in the degradation process. Deletion of peptide:N-glycanase (PNG1) stabilizes this association, a process accompanied by slowdown of 6Myc-Hmg2p degradation. Truncation of the unstructured C-terminal 23 amino acids of 6Myc-Hmg2p rendered its degradation PNG1-independent and allowed its partial delivery to the vacuole in an autophagy-dependent manner. These findings demonstrate a new conduit for the selective vacuolar/lysosomal removal of ERAD misfolded proteins by an autophagy-related machinery acting concomitantly with the proteasome.

CCZ1,MON1andYPT7genes are involved in pexophagy, the Cvt pathway and non-specific macroautophagy in the methylotrophic yeastPichia pastoris

Orthologues of Saccharomyces cerevisiae CCZ1, MON1 and YPT7 genes in the methylotrophic yeast, Pichia pastoris, have been identified. These genes encode proteins, which act as a complex, being involved in degradation of oleate-induced peroxisomes, Cvt (cytoplasm to vacuole targeting) pathway and non-specific macroautophagy in S. cerevisiae. CCZ1, MON1 and YPT7 gene orthologues are essential for multiple delivery pathways in P. pastoris. Strains with deletion of either of these genes displayed complete deficiency in pexophagy, non-specific macroautophagy and the biosynthetic Cvt pathway. The data suggest that CCZ1, MON1 and YPT7 genes are involved in degradation of both small oleate-induced and large methanol-induced peroxisomes. The data suggest conservative functions of CCZ1, MON1 and YPT7 genes among yeast species.

Differential roles for the low-affinity phosphate transporters Pho87 and Pho90 inSaccharomyces cerevisiae

When starved of P(i), yeast cells activate the PHO signalling pathway, wherein the Pho4 transcription factor mediates expression of genes involved in P(i) acquisition, such as PHO84, encoding the high-affinity H(+)/P(i) symporter. In contrast, transcription of PHO87 and PHO90, encoding the low-affinity H(+)/P(i) transport system, is independent of phosphate status. In the present work, we reveal that, upon P(i) starvation, these low-affinity P(i) transporters are endocytosed and targeted to the vacuole. For Pho87, this process strictly depends on SPL2, another Pho4-dependent gene that encodes a protein known to interact with the N-terminal SPX domain of the transporter. In contrast, the vacuolar targeting of Pho90 upon Pi starvation is independent of both Pho4 and Spl2, although it still requires its SPX domain. Furthermore, both Pho87 and Pho90 are also targeted to the vacuole upon carbon-source starvation or upon treatment with rapamycin, which mimics nitrogen starvation, but although these responses are independent of PHO pathway signalling, they again require the N-terminal SPX domain of the transporters. These observations suggest that other SPX-interacting proteins must be involved. In addition, we show that Pho90 is the most important P(i) transporter under high P(i) conditions in the absence of a high-affinity P(i)-transport system. Taken together, our results illustrate that Pho87 and Pho90 represent non-redundant P(i) transporters, which are tuned by the integration of multiple nutrient signalling mechanisms in order to adjust P(i)-transport capacity to the general nutritional status of the environment.

P62 targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)-associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.

Dissecting cardosin B trafficking pathways in heterologous systems

In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumulates in the extracellular matrix of the transmitting tissue. Given cardosins' high homology and yet different cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plasticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and transient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular trafficking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggesting that either the glycans are inaccessible to the Golgi processing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-modifying enzymes are able to modify the glycans. Concerning cardosin B trafficking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the differential targeting of cardosins A and B in cardoon pistils results principally from differences in the cells in which these two proteins are expressed.

Soybean peroxidase propeptides are functional signal peptides and increase the yield of a foreign protein

Elements that contribute to the high, stable yield of soybean peroxidase (SBP) in soybean seed coats can be exploited in the development of this tissue as a protein production platform. SBP contains an N-terminal and a C-terminal propeptide that are predicted to direct vacuolar targeting; this may be one factor that contributes to its high yield and stability. We characterized the function of the SBP propeptides and investigated their ability to increase the yield of a foreign protein in a heterologous plant system. SBP propeptides are functional signal peptides capable of directing vacuolar transport in Arabidopsis. The use of these propeptides as well as an endoplasmic reticulum (ER)-retention signal to direct a foreign protein to the apoplast, ER, or vacuole can significantly increase yield and will therefore be useful for the development of the seed coat as a protein production platform. We also demonstrate that growth conditions may have a significant impact on the yield of a foreign protein and that this may be subcellular compartment-specific.

Efficient ER Exit and Vacuole Targeting of Yeast Sna2p Require Two Tyrosine-Based Sorting Motifs

SNA (Sensitive to Na(+)) proteins form a membrane protein family, which, in the yeast Saccharomyces cerevisiae, is composed of four members: Sna1p/Pmp3p, Sna2p, Sna3p and Sna4p. In this study, we focused on the 79 residue Sna2p protein. We found that Sna2p is localized in the vacuolar membrane. Directed mutagenesis showed that two functional tyrosine motifs YXXØ are present in the C-terminal region. Each of these is involved in a different Golgi-to-vacuole targeting pathway: the tyrosine 65 motif is involved in adaptor protein (AP-1)-dependent targeting, whereas the tyrosine 75 motif is involved in AP-3-dependent targeting. Moreover, our data suggest that these motifs also play a crucial role in the exit of Sna2p from the endoplasmic reticulum (ER). Directed mutagenesis of these tyrosines led to a partial redirection of Sna2p to lipid bodies, probably because of a decrease in ER exit efficiency. Sna2p is the first yeast protein in which two YXXØ motifs have been identified and both were shown to be functional at two different steps of the secretory pathway, ER exit and Golgi-to-vacuole transport.

Daniel Klionsky: A full plate for autophagy

Klionsky proves that those who can, teach as well as do.

Characterization of Aspergillus nidulans DidBDid2, a non-essential component of the multivesicular body pathway

ESCRT-III heteropolymers mediate membrane protein cargo sorting into multivesicular endosomes for subsequent vacuolar degradation. We studied the localization of largely uncharacterized Aspergillus nidulans ESCRT-III using its key structural component Vps32 and the ‘associated’ component DidBDid2. Vps32-GFP localizes to motile early endosomes as reported, but predominates in aggregates often associated with vacuoles due to inability to dissociate from endosomes. DidBDid2 regulating Vps4 (the ATPase disassembling ESCRT-III) is not essential. Consistent with this accessory role, didBΔ is unable to block the MVB sorting of the glutamate transporter AgtA, but increases its steady-state level and mislocalizes a fraction of the permease to the plasma membrane under conditions promoting its vacuolar targeting. didBΔ exacerbates the dominant-negative growth defect resulting from Vps32-GFP over-expression. A proportion of DidB-GFP is detectable in early endosomes colocalizing with RabARab5 and accumulating in nudA1 tips, suggesting that ESCRT-III assembles on endosomes from the early steps of the endocytic pathway.

Roles of the Lipid-binding Motifs of Atg18 and Atg21 in the Cytoplasm to Vacuole Targeting Pathway and Autophagy

Atg18 and Atg21 are homologous WD-40 repeat proteins that bind phosphoinositides via a novel conserved Phe-Arg-Arg-Gly motif and function in autophagy-related pathways. Atg18 is required for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas Atg21 is only required for the Cvt pathway. Currently, the functions of both proteins are poorly understood. Here, we examined the relationship between the phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding abilities of Atg18 and Atg21 and autophagy by expressing variants of these proteins that have mutations in their phosphoinositide-binding motifs. Cells expressing PtdIns(3)P-binding mutants of both these proteins showed highly reduced autophagy. Furthermore, the localization of components of two related ubiquitin-like protein conjugation systems, Atg8 and Atg16, to the phagophore assembly site is affected. Consistent with the aberrant localization of the above Atg proteins, precursor Ape1, a cargo of the Cvt pathway and autophagy, is partially protease-sensitive in starvation conditions. This finding suggests a requirement for the PtdIns(3)P binding capability of Atg18 and Atg21 in efficient completion of the sequestering autophagic vesicles. Finally, using a multiple knock-out strain, we found that Atg18 and Atg21 facilitate the recruitment of Atg8-PE to the site of autophagosome formation and protect it from premature cleavage by Atg4, which represents a key aspect of post-translational autophagy regulation. Taken together, our results suggest that PtdIns(3)P binding by at least Atg18 or Atg21 is required for robust autophagic activity and that the PtdIns(3)P-binding motifs of Atg18 and Atg21 can compensate for one another in the recruitment of Atg components that are dependent on PtdIns(3)P for their phagophore assembly site association.

Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways

Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5;1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5;1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5;1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.

The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy

Macroautophagy is a catabolic pathway used for the turnover of long-lived proteins and organelles in eukaryotic cells. The morphological hallmark of this process is the formation of double-membrane autophagosomes that sequester cytoplasm. Autophagosome formation is the most complex part of macroautophagy, and it is a dynamic event that likely involves vesicle fusion to expand the initial sequestering membrane, the phagophore; however, essentially nothing is known about this process including the molecular components involved in vesicle tethering and fusion. In this study, we provide evidence that the subunits of the conserved oligomeric Golgi (COG) complex are required for double-membrane cytoplasm to vacuole targeting vesicle and autophagosome formation. COG subunits localized to the phagophore assembly site and interacted with Atg (autophagy related) proteins. In addition, mutations in the COG genes resulted in the mislocalization of Atg8 and Atg9, which are critical components involved in autophagosome formation.

Comparative efficiency of subcellular targeting signals for expression of a toxic protein in sugarcane

Transgenic sugarcane plants (Saccharum hybrid) have been proposed as a production platform for recombinant proteins, including those providing pathogen resistance as well as high value therapeutic proteins. For the in planta production of proteins that are potentially toxic, a careful consideration of subcellular location is required in order to optimise yield and to avoid detrimental interaction with plant cellular processes. In this study, avidin, a glycoprotein that is potentially toxic to cells because of its high affinity to the co-vitamin biotin, was used to test the effectiveness of a range of targeting signals. Accumulation of avidin was directed to the apoplast, endoplasmic reticulum and to the lytic and delta type vacuoles. Although targeting to the delta vacuole resulted in the highest yields of avidin, these plants developed a biotin deficient phenotype, indicating that this targeting was not fully effective in protecting cellular biotin pools. Similar problems were also observed when avidin was retained in the endoplasmic reticulum. When avidin was targeted to the lytic vacuole using the targeting signal from the sugarcane legumain, plants remained phenotypically normal; however, avidin was predominantly detected as a degraded product due to site-specific limited proteolysis in the vacuole. For avidin and other potentially toxic products, this lytic vacuole targeting signal may be useful if stability within this proteolytic environment can be improved.

Aup1-mediated Regulation of Rtg3 during Mitophagy

Mitophagy is an autophagic process that degrades mitochondria by an intracellular engulfment that leads to their delivery into the lumen of the cell's hydrolytic compartment, such as the lysosome in animal cells or the vacuole in yeast. It is hypothesized that such processes serve a quality control function to prevent or slow the accumulation of malfunctioning mitochondria, which are thought in turn to underlie central aspects of the aging process in eukaryotic organisms. We recently identified a conserved mitochondrial protein phosphatase homolog, Aup1, which is required for efficient stationary phase mitophagy in yeast. In the present report, we demonstrate that the retrograde signaling pathway (RTG) is defective in aup1Delta mutants. In agreement with a role for Aup1 in the regulation of the RTG pathway, we find that deletion of RTG3, a transcription factor that mediates the RTG response, causes a defect in stationary phase mitophagy and that deletion of AUP1 leads to changes in Rtg3 phosphorylation patterns under these conditions. In addition, we find that mitophagic conditions lead to induction of RTG pathway target genes in an Aup1-dependent fashion. Thus, our results suggest that the function of Aup1 in mitophagy could be explained through its regulation of Rtg3-dependent transcription.

Recombinant expression and characterization of Solanum tuberosum aspartic protease

Unique to aspartic proteinases from plants are the presence of approximately 100 amino acid regions, which are usually excised during activation of the zymogen. These sequences are termed ‘Plant-Specific Inserts’ and are implicated in membrane interactions of their parent enzymes, including vacuolar targeting and host defense. The need to further characterize the structure-function rol (s) of plant-specific inserts stimulated the current study of the characterization of Solanum tuberosum AP (StAP). Recombinant expression of wild-type StAP resulted in the 54 kDa protein being visualized by SDS-PAGE analysis with a protein yield of 0.03%. A protein purification factor could not be established since activation of the protein at pH 2.2, 3.0, 3.7 and 5.5 was not achieved as evidenced by a lack of change in band patterns on SDS-PAGE as well as acidification and hemoglobin degradation assays. To potentially improve enzyme folding and activation ability, two mutants, (1) lacking the pre-signal sequence and (2) lacking both the signal sequence and the prosegment, were designed, sub-cloned, and expressed. Both products proved to be insoluble and inactive. New constructs were designed for the expression of StAP inclusion bodies for insoluble expression and subsequent re-folding of the protein. Additionally, CysAla mutations for each PSI Cys residue were made to investigate the rol (s) of plant-specific insert disulfide bonds in plant aspartic proteinase enzyme folding and structure. All PSI cysteine mutations (eight mutants) were successfully created using QuickChange MutagenesisTM

Down-regulation of a manganese transporter in the face of metal toxicity.

The yeast Smf1p Nramp manganese transporter is posttranslationally regulated by environmental manganese. Smf1p is stabilized at the cell surface with manganese starvation, but is largely degraded in the vacuole with physiological manganese through a mechanism involving the Rsp5p adaptor complex Bsd2p/Tre1p/Tre2p. We now describe an additional level of Smf1p regulation that occurs with toxicity from manganese, but not other essential metals. This regulation is largely Smf1p-specific. As with physiological manganese, toxic manganese triggers vacuolar degradation of Smf1p by trafficking through the multivesicular body. However, regulation by toxic manganese does not involve Bsd2p/Tre1p/Tre2p. Toxic manganese triggers both endocytosis of cell surface Smf1p and vacuolar targeting of intracellular Smf1p through the exocytic pathway. Notably, the kinetics of vacuolar targeting for Smf1p are relatively slow with toxic manganese and require prolonged exposures to the metal. Down-regulation of Smf1p by toxic manganese does not require transport activity of Smf1p, whereas such transport activity is needed for Smf1p regulation by manganese starvation. Furthermore, the responses to manganese starvation and manganese toxicity involve separate cellular compartments. We provide evidence that manganese starvation is sensed within the lumen of the secretory pathway, whereas manganese toxicity is sensed within an extra-Golgi/cytosolic compartment of the cell.