Compartment acidification is required for efficient sorting of proteins to the vacuole in Saccharomyces cerevisiae.

The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates a pH gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of the genes encoding the A, B, or c subunit of the vacuolar ATPase are unable to acidify their vacuoles. These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles. The presence of mature forms of the vacuolar proteins within the cell indicates that vat mutations do not cause defects in zymogen processing. Precursor forms of the membrane-associated vacuolar hydrolase alkaline phosphatase are also accumulated in vat mutant cells but to a lesser extent, suggesting that sorting of vacuolar membrane proteins is less sensitive to changes in the lumenal pH. A similar type of missorting defect can be induced in wild-type cells at pH 7.5. These results indicate that acidification of the vacuolar system is important for efficient sorting of proteins to the vacuole.     

The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast

Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.     

Secretory bulk flow of soluble proteins is efficient and COPII dependent

COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley alpha-amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed.     

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.     

Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway

The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.     

A novel aspartic proteinase is targeted to the secretory pathway and to the vacuole in the moss Physcomitrella patens

In seed plants aspartic proteases (APs) are known to reside in storage vacuoles. Targeting to this compartment is provoked by a secretory signal peptide and the plant-specific insert (PSI). In order to study secretory and vacuolar targeting in a seedless plant, the moss Physcomitrella patens, we isolated a cDNA encoding PpAP1, a novel aspartic proteinase. Sequence alignment with other members of the family of plant APs (EC 3.4.23) revealed a high overall identity and the Pfam motifs for aspartic proteinase and PSI were clearly recognised. In phylogenetic analysis PpAP1 was placed at a very basal position outside of the bigger clusters. Protoplasts transiently expressing the PpAP1 signal peptide fused to GFP showed fluorescence in a well-developed ER-Golgi network. A C-terminal fusion of GFP to the entire PpAP1 protein showed vacuolar fluorescence in transiently transfected protoplasts. Therefore, the vacuole is apparently the in-vivo target for PpAP1. In this study the three-dimensional peculiarity of the endomembrane continuum of ER and Golgi was visualised in a seedless plant for the first time. Above all the functionality of the secretory and the vacuolar targeting signals make them become useful tools for biotechnological approaches.     

Whole-field integration, not detailed analysis, is used by the crab optokinetic system to separate rotation and translation in optic flow

For optimal visual control of compensatory eye movements during locomotion it is necessary to distinguish the rotational and translational components of the optic flow field. Optokinetic eye movements can reduce the rotational component only, making the information contained in the translational flow readily available to the animal. We investigated optokinetic eye rotation in the marble rock crab, Pachygrapsus marmoratus, during translational movement, either by displacing the animal or its visual surroundings. Any eye movement in response to such stimuli is taken as an indication that the system is unable to separate the translational and the rotational components in the optic flow in a mathematically perfect way. When the crabs are translated within a pseudo-natural environment, eye movements are negligible, especially during sideways translation. When, however, crabs were placed in a gangway between two elongated rectangular sidewalls carrying dotted patterns which were translated back and forth, marked eye movements were elicited, depending on the translational velocity. To resolve this discrepancy, we tested several hypotheses about mechanisms using detailed analysis of the optic flow or whole-field integration. We found that the latter are sufficient to explain the efficient separation of translation and rotation of crabs in quasi-natural situations. Accepted: 6 May 1997     

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl₂ to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by ³¹P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na⁺ and Mg²⁺ are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K⁺ is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg²⁺ and Na⁺, show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K⁺, the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.Abbreviation NMR nuclear magnetic resonanceThe very cooperative help by Dr. J. Reiner with the ³¹P-NMR measurements and the technical assistance by D. Keis are gratefully acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie.     

Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.     

Inhibition of the vacuolar H+-ATPase perturbs the transport, sorting, processing and release of regulated secretory proteins.

Vacuolar H+-ATPases (V-ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V-ATPase inhibitor bafilomycin A1 (Baf) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf-treated intermediate pituitary cells revealed a reduction in the amount of small dense-core secretory granules and the appearance of vacuolar structures in the trans-Golgi area. Pulse-chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found that treated cells secreted considerable amounts of a PC2 processing intermediate and unprocessed SgIII in a constitutive fashion. Collectively, these data indicate that in the secretory pathway, V-ATPases play an important role in creating the microenvironment that is essential for proper transport, sorting, processing and release of regulated secretory proteins.     

Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A

Potato virus X (PVX) has been used as an expression vector to target the green fluorescent protein (GFP) from the jellyfish Aequorea victoria to the endoplasmic reticulum (ER) of tobacco (Nicotiana clevelandii L.) leaves. Expression of free GFP resulted in strong cytoplasmic fluorescence with organelles being imaged in negative contrast. Translocation of GFP into the lumen of the ER was mediated by the use of the sporamin signal peptide. Retention of GFP in the ER was facilitated by the splicing of the ER retrieval/retention tetrapeptide, KDEL to the carboxy terminus of GFP. Fluorescence of GFP was restricted to a labile cortical network of ER tubules with occasional small lamellae and to streaming trans-vacuolar strands. Secretion of ER-targeted GFP was inhibited both by cold shock and low concentrations of the secretory inhibitor brefeldin A. However, both prolonged cold and prolonged incubation in brefeldin A resulted in the recovery of secretory capability. In leaves infected with the GFP-KDEL construct, high concentrations of brefeldin A induced the tubular network of cortical ER to transform into large lamellae or sheets which reverted to the tubular network on removal of the drug. Received: 8 October 1998 / Accepted: 16 November 1998     

Identification of the protein storage vacuole and protein targeting to the vacuole in leaf cells of three plant species

Protein storage vacuoles (PSVs) are specialized vacuoles devoted to the accumulation of large amounts of protein in the storage tissues of plants. In this study, we investigated the presence of the storage vacuole and protein trafficking to the compartment in cells of tobacco (Nicotiana tabacum), common bean (Phaseolus vulgaris), and Arabidopsis leaf tissue. When we expressed phaseolin, the major storage protein of common bean, or an epitope-tagged version of alpha-tonoplast intrinsic protein (alpha-TIP, a tonoplast aquaporin of PSV), in protoplasts derived from leaf tissues, these proteins were targeted to a compartment ranging in size from 2 to 5 microm in all three plant species. Most Arabidopsis leaf cells have one of these organelles. In contrast, from one to five these organelles occurred in bean and tobacco leaf cells. Also, endogenous alpha-TIP is localized in a similar compartment in untransformed leaf cells of common bean and is colocalized with transiently expressed epitope-tagged alpha-TIP. In Arabidopsis, phaseolin contained N-glycans modified by Golgi enzymes and its traffic was sensitive to brefeldin A. However, trafficking of alpha-TIP was insensitive to brefeldin A treatment and was not affected by the dominant-negative mutant of AtRab1. In addition, a modified alpha-TIP with an insertion of an N-glycosylation site has the endoplasmic reticulum-type glycans. Finally, the early step of phaseolin traffic, from the endoplasmic reticulum to the Golgi complex, required the activity of the small GTPase Sar1p, a key component of coat protein complex II-coated vesicles, independent of the presence of the vacuolar sorting signal in phaseolin. Based on these results, we propose that the proteins we analyzed are targeted to the PSV or equivalent organelle in leaf cells and that proteins can be transported to the PSV by two different pathways, the Golgi-dependent and Golgi-independent pathways, depending on the individual cargo proteins.     

Superantigen-induced T cell:B cell conjugation is mediated by LFA-1 and requires signaling through Lck, but not ZAP-70.

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.     

Atg23 is essential for the cytoplasm to vacuole targeting pathway and efficient autophagy but not pexophagy

Cells must regulate both biosynthesis and degradation to ensure proper homeostasis of cellular organelles and proteins. This balance is demonstrated in a unique way in the yeast Saccharomyces cerevisiae, which possesses two distinct, yet mechanistically related trafficking routes mediating the delivery of proteins from the cytoplasm to the vacuole: the biosynthetic cytoplasm to vacuole targeting (Cvt) and the degradative autophagy pathways. Several components employed by these two transport routes have been identified, but their mechanistic interactions remain largely unknown. Here we report a novel gene involved in these pathways, which we have named ATG23. Atg23 localizes to the pre-auto-phagosomal structure but also to other cytosolic punctate compartments. Our characterization of the Atg23 protein indicates that it is required for the Cvt pathway and efficient autophagy but not pexophagy. In the absence of Atg23, cargo molecules such as prApe1 are correctly recruited to a pre-autophagosomal structure that is unable to give rise to Cvt vesicles. We also demonstrate that Atg23 is a peripheral membrane protein that requires the presence of Atg9/Apg9 to be specifically targeted to lipid bilayers. Atg9 transiently interacts with Atg23 suggesting that it participates in the recruitment of this protein.     

Dibasic cleavage site is required for sorting to the regulated secretory pathway for both pro- and neuropeptide Y

To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non-mutated proneuropeptide Y (ProNPY) in pituitary-derived AtT20 cells. The mutations were carried out on dibasic cleavage site and or ProNPY C-terminal sequence. Targeting to the regulated secretory pathway was studied using protein kinase A (8-BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase. The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive-like secretory pathways. ProNPY secretion behaviour was not Proteolytic cleavage efficiency-dependent. The dibasic cleavage was essential for ProNPY and NPY cAMP-dependent regulated secretion and may have function as a retention signal.     

Transport of LAPTM5 to lysosomes requires association with the ubiquitin ligase Nedd4, but not LAPTM5 ubiquitination

LAPTM5 is a lysosomal transmembrane protein expressed in immune cells. We show that LAPTM5 binds the ubiquitin-ligase Nedd4 and GGA3 to promote LAPTM5 sorting from the Golgi to the lysosome, an event that is independent of LAPTM5 ubiquitination. LAPTM5 contains three PY motifs (L/PPxY), which bind Nedd4-WW domains, and a ubiquitin-interacting motif (UIM) motif. The Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the LAPTM5-UIM; this interaction does not require the GGA3-GAT domain. LAPTM5 mutated in its Nedd4-binding sites (PY motifs) or its UIM is retained in the Golgi, as is LAPTM5 expressed in cells in which Nedd4 or GGA3 is knocked-down with RNAi. However, ubiquitination-impaired LAPTM5 can still traffic to the lysosome, suggesting that Nedd4 binding to LAPTM5, not LAPTM5 ubiquitination, is required for targeting. Interestingly, Nedd4 is also able to ubiquitinate GGA3. These results demonstrate a novel mechanism by which the ubiquitin-ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination.     

Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB

Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.     

Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles

Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seeds. These vesicles were designated precursor-accumulating (PAC) vesicles and had diameters of 200 to 400 nm. They contained an electron-dense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained small vesicle-like structures. Immunocytochemical analysis revealed numerous electron-dense aggregates of storage proteins within the endoplasmic reticulum. It is likely that these aggregates develop into the electron-dense cores of the PAC vesicles and then leave the endoplasmic reticulum. Immunocytochemical analysis also showed that complex glycans are associated with the peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These results suggest that the unique PAC vesicles might mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles.     

The C-terminal extension of a hybrid immunoglobulin A/G heavy chain is responsible for its Golgi-mediated sorting to the vacuole

We have assessed the ability of the plant secretory pathway to handle the expression of complex heterologous proteins by investigating the fate of a hybrid immunoglobulin A/G in tobacco cells. Although plant cells can express large amounts of the antibody, a relevant proportion is normally lost to vacuolar sorting and degradation. Here we show that the synthesis of high amounts of IgA/G does not impose stress on the plant secretory pathway. Plant cells can assemble antibody chains with high efficiency and vacuolar transport occurs only after the assembled immunoglobulins have traveled through the Golgi complex. We prove that vacuolar delivery of IgA/G depends on the presence of a cryptic sorting signal in the tailpiece of the IgA/G heavy chain. We also show that unassembled light chains are efficiently secreted as monomers by the plant secretory pathway.