Sorting of plant vacuolar proteins is initiated in the ER

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor–ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer‐mediated recycling of the plant VSR BP80 starts at the trans‐Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co‐expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII‐mediated transport route. Retention of soluble cargo despite ongoing COPII‐mediated bulk flow can only be explained by an interaction with membrane‐bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER‐anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR–ligand interaction. It also implies that the retromer‐mediated recycling route for the VSRs leads from the TGN back to the ER.     

Arv1 Regulates PM and ER Membrane Structure and Homeostasis But is Dispensable for Intracellular Sterol Transport

The pan‐eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild‐type and Arv1‐deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport‐coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild‐type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C‐terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail‐anchored proteins involved in membrane homoeostasis .     

Immunogold localization of prolamines in developingHaynaldia villosa endosperm

Summary Haynaldia villosa is a wild grass belonging to the tribe Triticeae, which includes important crops such as wheat, barley, and rye. The alcohol-soluble proteins ofH. villosa have extensive immunological relatedness with wheat prolamines as visualized by Western blot analysis. Amorphous protein inclusions surrounded by a limiting membrane are commonly found in the vacuoles of endosperm and subaleurone layers ofH. villosa seeds. A layer of cells just beneath the aleurone layer is rich in ER. Unlike that in other cell types, the ER in these cells is highly dilated and contains materials at its swollen distal ends. These materials are structurally similar to substances found in the protein bodies. Protein A-gold immunocytochemical localization studies employing antibodies against wheat prolamine confirmed that the inclusions found in the lumen of the ER do not contain prolamines. This observation indicates that the ER does not act as the site of prolamine accumulation inH. villosa. Protein bodies found in the vacuoles and the vesicles associated with the Golgi complexes were specifically labeled. This suggests that Golgi complexes mediate the transport of prolamines into vacuoles ofH. villosa endosperm cells, in a fashion analogous to that of other vacuolar proteins of dicotyledonous plants.     

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi.     

V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

Genome-wide Analysis of AP-3–dependent Protein Transport in Yeast

The evolutionarily conserved adaptor protein-3 (AP-3) complex mediates cargo-selective transport to lysosomes and lysosome-related organelles. To identify proteins that function in AP-3–mediated transport, we performed a genome-wide screen in Saccharomyces cerevisiae for defects in the vacuolar maturation of alkaline phosphatase (ALP), a cargo of the AP-3 pathway. Forty-nine gene deletion strains were identified that accumulated precursor ALP, many with established defects in vacuolar protein transport. Maturation of a vacuolar membrane protein delivered via a separate, clathrin-dependent pathway, was affected in all strains except those with deletions of YCK3, encoding a vacuolar type I casein kinase; SVP26, encoding an endoplasmic reticulum (ER) export receptor for ALP; and AP-3 subunit genes. Subcellular fractionation and fluorescence microscopy revealed ALP transport defects in yck3Δ cells. Characterization of svp26Δ cells revealed a role for Svp26p in ER export of only a subset of type II membrane proteins. Finally, ALP maturation kinetics in vac8Δ and vac17Δ cells suggests that vacuole inheritance is important for rapid generation of proteolytically active vacuolar compartments in daughter cells. We propose that the cargo-selective nature of the AP-3 pathway in yeast is achieved by AP-3 and Yck3p functioning in concert with machinery shared by other vacuolar transport pathways.     

Nonpolarized cells selectively sort apical proteins from cell surface to a novel compartment,but lack apical retention mechanisms

Membrane trafficking is central to establishing and maintaining epithelial cell polarity. One open question is to what extent the mechanisms regulating membrane trafficking are conserved between nonpolarized and polarized cells. To answer this question, we examined the dynamics of domain-specific plasma membrane (PM) proteins in three classes of hepatic cells: polarized and differentiated WIF-B cells, nonpolarized and differentiated Fao cells, and nonpolarized and nondifferentiated Clone 9 cells. In nonpolarized cells, mature apical proteins were uniformly distributed in the PM. Surprisingly, they were also in an intracellular compartment. Double labeling revealed that the compartment contained only apical proteins. By monitoring the dynamics of antibody-labeled molecules in nonpolarized cells, we further found that apical proteins rapidly recycled between the compartment and PM. In contrast, the apical PM residents in polarized cells showed neither internalization nor return to the basolateral PM from which they had originally come. Cytochalasin D treatment of these polarized cells revealed that the retention mechanisms are actin dependent. We conclude from these data that both polarized and nonpolarized cells selectively sort apical proteins from the PM and transport them to specific, but different cellular locations. We propose that the intracellular recycling compartment in nonpolarized cells is an intermediate in apical surface formation.     

BsdABsd2‐dependent vacuolar turnover of a misfolded version of the UapA transporter along the secretory pathway: prominent role of selective autophagy

Transmembrane proteins translocate cotranslationally in the endoplasmic reticulum (ER) membrane and traffic as vesicular cargoes, via the Golgi, in their final membrane destination. Misfolding in the ER leads to protein degradation basically through the ERAD/proteasome system. Here, we use a mutant version of the purine transporter UapA (ΔR481) to show that specific misfolded versions of plasma membrane cargoes undergo vacuolar turnover prior to localization in the plasma membrane. We show that non‐endocytic vacuolar turnover of ΔR481 is dependent on BsdA^Bsd2, an ER transmembrane adaptor of HulA^Rsp5 ubiquitin ligase. We obtain in vivo evidence that BsdA^Bsd2 interacts with HulA^Rsp5 and ΔR481, primarily in the ER. Importantly, accumulation of ΔR481 in the ER triggers delivery of the selective autophagy marker Atg8 in vacuoles along with ΔR481. Genetic block of autophagy (atg9Δ, rabO^ts) reduces, but does not abolish, sorting of ΔR481 in the vacuoles, suggesting that a fraction of the misfolded transporter might be redirected for vacuolar degradation via the Golgi. Our results support that multiple routes along the secretory pathway operate for the detoxification of Aspergillus nidulans cells from misfolded membrane proteins and that BsdA is a key factor for marking specific misfolded cargoes.     

Antibody to AP1B adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit micro1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3-5 min. By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in approximately 45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.     

Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae

Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins. To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p. Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells. Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker. Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin beta-lactone. Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment. Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p. Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation. These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae.     

An engineered C‐terminal disulfide bond can partially replace the phaseolin vacuolar sorting signal

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N‐terminal half of the Zea mays prolamin γ‐zein forms interchain disulfide bonds that facilitate the formation of ER‐located protein bodies. Wild‐type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C‐terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Δ418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild‐type protein. Moreover, Δ418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.     

Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'- diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D- labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.     

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.     

Intrinsically Disordered Linker and Plasma Membrane‐Binding Motif Sort Ist2 and Ssy1 to Junctions

Membrane junctions or contact sites are close associations of lipid bilayers of heterologous organelles. Ist2 is an endoplasmic reticulum (ER)‐resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker's yeast. We asked the question what structure in Ist2 bridges the up to 30 nm distance between the PM and the cER and we noted that the region spacing the transmembrane domain from the cortical sorting signal interacting with the PM is predicted to be intrinsically disordered (ID). In Ssy1, a protein that was not previously described to reside at membrane junctions, we recognized a domain organization similar to that in Ist2. We found that the localization of both Ist2 and Ssy1 at the cell periphery depends on the presence of a PM‐binding domain, an ID linker region of sufficient length and a transmembrane domain that most probably resides in the ER. We show for the first time that an ID amino acid domain bridges adjacent heterologous membranes. The length and flexibility of ID domains make them uniquely eligible for spanning large distances, and we suggest that this domain structure occurs more frequently in proteins that mediate the formation of membrane contact sites.      

ACAP1 promotes endocytic recycling by recognizing recycling sorting signals

Cargo sorting that promotes the transport of cargo proteins from a membrane compartment has been predicted to be unlikely in the endocytic recycling pathways. We now show that ACAP1 binds specifically and directly to recycling cargo proteins. Reducing this interaction for TfR inhibits its recycling. Moreover, ACAP1 binds to two distinct phenylalanine-based sequences in the cytoplasmic domain of TfR that function as recycling sorting signals to promote its transport from the recycling endosome. Taken together, these findings indicate that ACAP1 promotes cargo sorting by recognizing recycling sorting signals.     

Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane

The Vps1 protein of Saccharomyces cerevisiae is an 80-kD GTPase associated with the Golgi apparatus. Vps1p appears to play a direct role in the retention of late Golgi membrane proteins, which are mislocalized to the vacuolar membrane in its absence. The pathway by which late Golgi and vacuolar membrane proteins reach the vacuole in vps1 delta mutants was investigated by analyzing transport of these proteins in vps1 delta cells that also contained temperature sensitive mutations in either the SEC4 or END4 genes, which are required for a late step in secretion and the internalization step of endocytosis, respectively. Not only was vacuolar transport of a Golgi membrane protein blocked in the vps1 delta sec4-ts and vps1 delta end4-ts double mutant cells at the non-permissive temperature but vacuolar delivery of the vacuolar membrane protein, alkaline phosphatase was also blocked in these cells. Moreover, both proteins expressed in the vps1 delta end4- ts cells at the elevated temperature could be detected on the plasma membrane by a protease digestion assay indicating that these proteins are transported to the vacuole via the plasma membrane in vps1 mutant cells. These data strongly suggest that a loss of Vps1p function causes all membrane traffic departing from the late Golgi normally destined for the prevacuolar compartment to instead be diverted to the plasma membrane. We propose a model in which Vps1p is required for formation of vesicles from the late Golgi apparatus that carry vacuolar and Golgi membrane proteins bound for the prevacuolar compartment.     

Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant

The sec18 and sec23 secretory mutants of Saccharomyces cerevisiae have previously been shown to exhibit temperature-conditional defects in protein transport from the ER to the Golgi complex (Novick, P., S. Ferro, and R. Schekman, 1981. Cell. 25:461-469). We have found that the Sec18 and Sec23 protein functions are rapidly inactivated upon shifting mutant cells to the nonpermissive temperature (less than 1 min). This has permitted an analysis of the potential role these SEC gene products play in transport events distal to the ER. The sec-dependent transport of alpha-factor (alpha f) and carboxypeptidase Y (CPY) biosynthetic intermediates present throughout the secretory pathway was monitored in temperature shift experiments. We found that Sec18p/NSF function was required sequentially for protein transport from the ER to the Golgi complex, through multiple Golgi compartments and from the Golgi complex to the cell surface. In contrast, Sec23p function was required in the Golgi complex, but only for transport of alpha f out of an early compartment. Together, these studies define at least three functionally distinct Golgi compartments in yeast. From cis to trans these compartments contain: (a) An alpha 1----6 mannosyltransferase; (b) an alpha 1----3 mannosyltransferase; and (c) the Kex2 endopeptidase. Surprisingly, we also found that a pool of Golgi-modified CPY (p2 CPY) located in a compartment distal to the alpha 1----3 mannosyltransferase does not require Sec18p function for final delivery to the vacuole. This compartment appears to be equivalent to the Kex2 compartment as we show that a novel vacuolar CPY-alpha f-invertase fusion protein undergoes efficient Kex2-dependent cleavage resulting in the secretion of invertase. We propose that this Kex2 compartment is the site in which vacuolar proteins are sorted from proteins destined to be secreted.     

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.     

Vacuolar degradation of two integral plasma membrane proteins, AtLRR84A and OsSCAMP1, is cargo ubiquitination-independent and prevacuolar compartment-mediated in plant cells

In plant cells, how integral plasma membrane (PM) proteins are degraded in a cargo ubiquitination-independent manner remains elusive. Here, we studied the degradative pathway of two plant PM proteins: AtLRR84A, a type I integral membrane protein belonging to the leucine-rich repeat receptor-like kinase protein family, and OsSCAMP1 (rice secretory carrier membrane protein 1), a tetraspan transmembrane protein located on the PM and trans-Golgi network (TGN) or early endosome (EE). Using wortmannin and ARA7(Q69L) mutant that could enlarge the multivesicular body (MVB) or prevacuolar compartment (PVC) as tools, we demonstrated that, when expressed as green fluorescent protein (GFP) fusions in tobacco BY-2 or Arabidopsis protoplasts, both AtLRR84A and OsSCAMP1 were degraded in the lytic vacuole via the internal vesicles of MVB/PVC in a cargo ubiquitination-independent manner. Such MVB/PVC-mediated vacuolar degradation of PM proteins was further supported by immunocytochemical electron microscopy (immunoEM) study showing the labeling of the fusions on the internal vesicles of the PVC/MVB. Thus, cargo ubiquitination-independent and PVC-mediated degradation of PM proteins in the vacuole is functionally operated in plant cells.     

Chlorophyll Biosynthetic Reactions during Senescence of Excised Barley (Hordeum vulgare L. cv IB 65) Leaves

The inhibitor sensitivity of the endoplasmic reticulum (ER) and plasma membrane (PM) calcium pumps of red beet (Beta vulgaris L.) were studied by measuring the ATP-driven accumulation of 45Ca2+ into isolated membrane vesicles. Both transporters were strongly inhibited by 50 [mu]mol m-3 erythrosin B, but only by 50% in the presence of 100 mmol m-3 vanadate. A number of inhibitors considered to be specific for the sarcoplasmic reticulum (SR)/ER-type calcium pump in animal cells were used to further characterize the PM and ER Ca2+-ATPases in red beet and were compared with their effect on the transport and hydrolytic activities of the PM and tonoplast H+-ATPases. The hydroquinones 2,5-di(tert-butyl)-1,4-benzohydroquinone and 2,5-di(tert-amyl)-1,4-benzohydroquinone produced around 20 and 40% inhibition of activity, respectively, of the PM and ER calcium pumps and the PM H+-ATPase when present at concentrations of 30 mmol m-3. In contrast, the vacuolar proton pump displayed a much higher sensitivity to these two compounds. Nonylphenol appeared to have a general inhibitory effect on all four membrane transport proteins and gave almost complete inhibition when present at a concentration of 100 mmol m-3. Thapsigargin and the structurally related compound trilobolide produced 50% inhibition of both the ER and PM calcium pumps at concentrations of 12.5 and 24 mmol m-3, respectively. The PM and tonoplast proton pumps were also sensitive to these compounds. The ER and PM calcium pumps were almost completely insensitive to cyclopiazonic acid (CPA) up to a concentration of 20 mmol m-3. When present at 100 mmol m-3 CPA caused 30% inhibition of the transport properties of all four ATPases. The high concentrations of all of the inhibitors of the SR/ER Ca-ATPase required to inhibit the red beet ER calcium pump, together with the similar effects on the PM calcium pump and the PM and tonoplast proton pumps, suggests that these hydrophobic compounds have a general nonselective action in red beet, possibly through disruption of membrane lipid-protein interactions.