The cargo in vacuolar storage protein transport vesicles is stratified

Developing pea seeds contain two functionally distinct vacuoles--lytic vacuoles and protein storage vacuoles (PSV). The Golgi apparatus of these cells has to discriminate between proteins destined for these vacuolar compartments. Whereas it is known that sorting into the lytic vacuole is performed via the conserved clathrin-coated vesicle pathway, sorting of proteins into the protein storage vacuole remains enigmatic. In developing pea cotyledons, the major storage proteins are sorted via 'dense vesicles'. In this report we examined the sorting of a minor protein of the protein storage vacuole, the sucrose-binding-protein homolog (SBP), along the secretory pathway employing immunoelectron microscopy on cryosectioned pea cotyledons. SBP follows the same vesicular route into the PSV as the main storage proteins legumin and vicilin, via the dense-vesicles. Furthermore, legumin and SBP are sorted together into the same dense vesicle population at the stack. Although soluble cargo proteins of the dense vesicles, they show a stratified distribution in the lumen of the dense vesicles. Whereas the legumin label is equally distributed across the lumen, the SBP label is concentrated at the membrane of the vesicle. This observation is discussed with respect to a putative receptor-mediated sorting of the proteins into the dense vesicles.     

Tonoplast intrinsic protein isoforms as markers for vacuolar functions

Plant cell vacuoles may have storage or lytic functions, but biochemical markers specific for the tonoplasts of functionally distinct vacuoles are poorly defined. Here, we use antipeptide antibodies specific for the tonoplast intrinsic proteins alpha-TIP, gamma-TIP, and delta-TIP in confocal immunofluorescence experiments to test the hypothesis that different TIP isoforms may define different vacuole functions. Organelles labeled with these antibodies were also labeled with antipyrophosphatase antibodies, demonstrating that regardless of their size, they had the expected characteristics of vacuoles. Our results demonstrate that the storage vacuole tonoplast contains delta-TIP, protein storage vacuoles containing seed-type storage proteins are marked by alpha- and delta- or alpha- and delta- plus gamma-TIP, whereas vacuoles storing vegetative storage proteins and pigments are marked by delta-TIP alone or delta- plus gamma-TIP. In contrast, those marked by gamma-TIP alone have characteristics of lytic vacuoles, and results from other researchers indicate that alpha-TIP alone is a marker for autophagic vacuoles. In root tips, relatively undifferentiated cells that contain vacuoles labeled separately for each of the three TIPs have been identified. These results argue that plant cells have the ability to generate and maintain three separate vacuole organelles, with each being marked by a different TIP, and that the functional diversity of the vacuolar system may be generated from different combinations of the three basic types.     

Protein sorting to the storage vacuoles of plants: a critical appraisal

The vacuole of plant cells is no longer considered to be a single compartment with multifunctional properties. A lot of evidence now points to the presence of multiple functionally distinct vacuolar compartments, some existing side by side in the same cell. As a consequence, the plant Golgi apparatus is faced with the problem of recognizing proteins destined for lytic and storage vacuoles and segregating them individually from the flow of secretory proteins to the cell surface. In contrast to acid hydrolases, which are sorted by BP-80-like receptors at the trans-Golgi of plant cells, the identification of receptors for storage proteins has in many ways resembled 'the search for the Holy Grail'. There are several candidates for storage protein receptors, but in no single case is the evidence entirely convincing. Much of the problem lies in the lack of consensus, sorting sequences in the proteins investigated. Other difficulties stem from 'out-of-context' heterologous expression studies. Evidence is now accumulating for the participation of hydrophobic sequences in inducing the formation of protein aggregates in the early Golgi apparatus, for which classical sorting receptors do not appear to be necessary. This review critically examines the current situation and contrasts the differences between data obtained in situ and data obtained transgenically. It highlights the so-called 'dense-vesicle' pathway and culminates with a discussion on the hitherto neglected problem of the intracellular transport of storage protein processing enzymes.     

The phaseolin vacuolar sorting signal promotes transient, strong membrane association and aggregation of the bean storage protein in transgenic tobacco

Vacuolar storage proteins of the 7S class are co-translationally introduced into the endoplasmic reticulum and reach storage vacuoles via the Golgi complex and dense vesicles. The signal for vacuolar sorting of one of these proteins, phaseolin of Phaseolus vulgaris, consists of a four-amino acid hydrophobic propeptide at the C-terminus. When this sequence is deleted, phaseolin is secreted instead of being sorted to vacuoles. It is shown here that in transgenic tobacco plants newly-synthesized phaseolin has unusual affinity to membranes and forms SDS-resistant aggregates, but mutated phaseolin polypeptides that are either secreted or defective in assembly do not have these characteristics. Association to membranes and aggregation are transient events: phaseolin accumulated in vacuoles is soluble in the absence of detergents and is not aggregated. Association to membranes starts before the phaseolin glycan acquires a complex structure and therefore before the protein reaches the medial or trans-cisternae of the Golgi complex. These results support the hypothesis of a relationship between aggregation and vacuolar sorting of phaseolin and indicate that sorting may start in early compartments of the secretory pathway.     

Sorting of proteins to storage vacuoles: how many mechanisms?

Vacuoles receive their proteins through the secretory pathway, this requires protein sorting signals and molecular machineries that, until recently, have been believed to be markedly distinct for lytic and storage vacuoles. However, new biochemical, morphological and genetic data indicate that the only known class of vacuolar sorting receptors, believed to be specific for lytic vacuoles, might also be involved in the sorting of certain storage proteins. Furthermore, storage vacuoles can have a complex multimembrane structure that is difficult to explain based on a single trafficking mechanism. A new array of possible molecular interactions is thus emerging that no longer supports a clear-cut distinction between the two types of vacuoles based on sorting signals and putative receptors.     

The protein storage vacuole: a unique compound organelle.

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.     

The Florey Lecture, 1992. The secretion of proteins by cells.

In eukaryotic cells, protein secretion provides a complex organizational problem. Secretory proteins are first transported, in an unfolded state, across the membrane of the endoplasmic reticulum (ER), and are then carried in small vesicles to the Golgi apparatus and finally to the cell membrane. The ER contains soluble proteins which catalyse the folding of newly synthesized polypeptides. These proteins are sorted from secretory proteins in the Golgi complex: they carry a sorting signal (the tetrapeptide KDEL or a related sequence) that allows them to be selectively retrieved and returned to the ER. This retrieval process also appears to be used by some bacterial toxins to aid their invasion of the cell: these toxins contain KDEL-like sequences and may, in effect, follow the secretory pathway in reverse. The membrane-bound receptor responsible for sorting luminal ER proteins has been identified in yeast by genetic means, and related receptors are found in mammalian cells. Unexpectedly, this receptor has a second role: in yeast it is required to maintain the normal size and function of the Golgi apparatus. By helping to maintain the composition of both ER and Golgi compartments, the KDEL receptor has an important role in the organization of the secretory pathway.     

Binding of BiP to an assembly-defective protein in plant cells

The binding protein (BiP) has been implicated as a mediator of protein folding and assembly in the endoplasmic reticulum of mammalian cells and has often been found in stable association with structurally defective proteins. To acquire information on the activity of BiP in plant cells, we have expressed in tobacco protoplasts the wild type form and an assembly-defective form of bean phaseolin. Phaseolin (PHSL) is a soluble, trimeric, storage glycoprotein co-translationally inserted into the lumen of the endoplasmic reticulum and then transported along the secretory pathway to the protein storage vacuoles. We have previously shown that a PHSL mutant in which the last 59 amino acids have been deleted (Δ363PHSL) is unable to form trimers and is retained in a pre-Golgi compartment when synthesized in Xenopus oocytes. When transiently expressed in tobacco leaf protoplasts, wild-type PHSL is correctly glycosylated and assembles efficiently and rapidly into trimers. Δ363PHSL is also correctly glycosylated but does not trimerize. Tobacco BiP and Δ363PHSL are co-immunoselected using either anti-PHSL or anti-BiP antibodies. Under the same conditions, co-immunoselection of BiP with wild-type PHSL is not detectable. The BiP bound to Δ363PHSL can be released by treatment of the complex with ATP, indicating that the binding is related to the proposed function of BiP in protein folding and assembly in the endoplasmic reticulum. These data indicate that BiP stably binds structurally defective proteins in plant cells.     

Cis-elements of protein transport to the plant vacuoles

Vacuolar proteins are synthesized and translocated into the endoplasmic reticulum and transported to the vacuoles through the secretory pathway. Three different types of vacuolar sorting signals have been identified, carried by N- or C-terminal propeptides or internal sequences. These signals are needed to target proteins to the different types of vacuoles that can coexist in a single plant cell. A conserved motif (NPIXL or NPIR) was identified within N-terminal propeptides, but can also function in a C-terminal propeptide and targets proteins in a receptor-mediated manner to a lytic vacuole. Binding to a family of putative sorting receptors for sequence-specific vacuolar sorting signals has been used as an assay to identify further peptides with other binding motifs. No motif was found in C-terminal sorting sequences, which need an accessible terminus, suggesting that they are recognized from the end by a still unknown receptor. The phosphatidylinositol kinase inhibitor wortmannin differentially affects sorting mediated by these two sorting sequences, suggesting different sorting mechanisms. Less is known about sorting mediated by internal protein sequences, which do not contain the conserved motif identified in N-terminal propeptides and by function by aggregation, leading to transport by coat-less dense vesicles to protein storage vacuoles. Even less is known about the sorting of tonoplast proteins, for which several sorting systems will also be needed.     

Plant RMR proteins: unique vacuolar sorting receptors that couple ligand sorting with membrane internalization

In receptor-mediated sorting of soluble protein ligands in the endomembrane system of eukaryotic cells, three completely different receptor proteins for mammalian (mannose 6-phosphate receptor), yeast (Vps10p) and plant cells (vacuolar sorting receptor; VSR) have in common the features of pH-dependent ligand binding and receptor recycling. In striking contrast, the plant receptor homology-transmembrane-RING-H2 (RMR) proteins serve as sorting receptors to a separate type of vacuole, the protein storage vacuole, but do not recycle, and their trafficking pathway results in their internalization into the destination vacuole. Even though plant RMR proteins share high sequence similarity with the best-characterized mammalian PA-TM-RING family proteins, these two families of proteins appear to play distinctly different roles in plant and animal cells. Thus, this minireview focuses on this unique sorting mechanism and traffic of RMR proteins via dense vesicles in various plant cell types.     

ESCRT-dependent vacuolar sorting and degradation of the auxin biosynthetic enzyme YUC1 flavin monooxygenase

YUC flavin monooxygenases catalyze the ratelimiting step of auxin biosynthesis. Here we report the vacuolar targeting and degradation of GFP-YUC1. GFP-YUC1 fusion expressed in Arabidopsis protoplasts or transgenic plants was primarily localized in vacuoles. Surprisingly, we established that GFP-YUC1, a soluble protein, was sorted to vacuoles through the ESCRT pathway, which has long been recognized for sorting and targeting integral membrane proteins. We further show that GFP-YUC1 was ubiquitinated and in this form GFP-YUC1 was targeted for degradation, a process that was also stimulated by elevated auxin levels. Our findings revealed a molecular mechanism of GFP-YUC1 degradation and demonstrate that the ESCRT pathway can recognize both soluble and integral membrane proteins as cargoes.     

The riddle of the plant vacuolar sorting receptors

Proteins synthesized on membrane-bound ribosomes are sorted at the Golgi apparatus level for delivery to various cellular destinations: the plasma membrane or the extracellular space, and the lytic vacuole or lysosome. Sorting involves the assembly of vesicles, which preferentially package soluble proteins with a common destination. The selection of proteins for a particular vesicle type involves the recognition of proteins by specific receptors, such as the vacuolar sorting receptors for vacuolar targeting. Most eukaryotic organisms have one or two receptors to target proteins to the lytic vacuole. Surprisingly, plants have several members of the same family, seven in Arabidopsis thaliana. Why do plants have so many proteins to sort soluble proteins to their respective destinations? The presence of at least two types of vacuoles, lytic and storage, seems to be a partial answer. In this review we analyze the last experimental evidence supporting the presence of different subfamilies of plant vacuolar sorting receptors.     

Spatial segregation of the regulated and constitutive secretory pathways

Recent experiments using DNA transfection have shown that secretory proteins in AtT-20 cells are sorted into two biochemically distinct secretory pathways. These two pathways differ in the temporal regulation of exocytosis. Proteins secreted by the regulated pathway are stored in dense-core granules until release is stimulated by secretagogues. In contrast, proteins secreted by the constitutive pathway are exported continuously, without storage. It is not known whether there are mechanisms to segregate regulated and constitutive secretory vesicles spatially. In this study, we examined the site of insertion of constitutive vesicles and compared it with that of regulated secretory granules. Regulated granules accumulate at tips of processes in these cells. To determine whether constitutively externalized membrane proteins are inserted into plasma membrane at the cell body or at process tips, AtT-20 cells were infected with ts-O45, a temperature-sensitive mutant of vesicular stomatitis virus in which transport of the surface glycoprotein G is conditionally blocked in the ER. After switching to the permissive temperature, insertion of G protein was detected at the cell body, not at process tips. Targeting of constitutive and regulated secretory vesicles to distinct areas of the plasma membrane appears to be mediated by microtubules. We found that while disruption of microtubules by colchicine had no effect on constitutive secretion, it completely blocked the accumulation of regulated granules at special release sites. Colchicine also affected the proper packaging of regulated secretory proteins. We conclude that regulated and constitutive secretory vesicles are targeted to different areas of the plasma membrane, most probably by differential interactions with microtubules. These results imply that regulated secretory granules may have unique membrane receptors for selective attachment to microtubules.     

Differential sorting and post-secretory targeting of proteins in parasitic invasion

Toxoplasma gondii uses a highly coordinated arsenal of three structurally and biochemically distinct secretory granules to invade and develop in a wide range of host cells. Proteins of these secretory granules are sorted to strategic subcellular locations using distinctive sorting signals and are then triggered differentially for exocytosis. These secreted proteins are subsequently targeted and inserted into membrane domains.     

Search and destroy: ER quality control and ER-associated protein degradation

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms.The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.     

Differential trafficking of soluble and integral membrane secretory granule-associated proteins

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.     

Renin is sorted to the regulated secretory pathway in transfected PC12 cells by a mechanism which does not require expression of the pro-peptide

The rat pheochromocytoma cell line PC12 targets secretory proteins into two distinct pathways. When DNA encoding human prorenin was transfected into PC12 cells, the protein was sorted into the regulated secretory pathway and released with similar kinetics to noradrenaline upon carbachol stimulation. To determine whether information for targeting prorenin lies within the pro-peptide we have transfected PC12 cells with a construct lacking the pro-peptide coding sequence. The transformed line secretes an apparently fully active enzyme and responds to carbachol stimulation with a rapid release of renin activity. We conclude that the pro-peptide of renin is not essential for targeting the protein to the regulated pathway in PC12 cells.     

Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways

Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.     

Search and Destroy: ER Quality Control and ER-Associated Protein Degradation

Abstract

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms. The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.