The plant cell vacuole has multiple functions, including storage of proteins and maintenance of an acidic pH where proteases will have maximal activity. It has been assumed that these diverse functions occur in the same compartment. Here, we demonstrate that antibodies to two different tonoplast intrinsic proteins, alpha-TIP and TIP-Ma27, label vacuole membranes of two different compartments within the same cell. These compartments are functionally distinct, because barley lectin, a protein stored in root tips, is exclusively contained within the alpha-TIP compartment, while aleurain, a protease that serves as a marker for an acidified vacuolar environment, is exclusively contained within the TIP-Ma27 compartment. As cells develop large vacuoles, the two compartments merge; this may represent a process by which storage products in the alpha-TIP compartment are exposed to the acidic lytic TIP-Ma27 compartment for degradation.

Clathrin-coated vesicles are known to be involved in the transport ofproteins from the Golgi to the vacuole in plant cells. The mechanisms by which proteins are directed into this pathway are not known. Here we identify an integral membrane protein of -80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum saivum L.) cotyledons, that bound at neutral pH to an affinity column prepared with the N-terminal targeting determinant of the vacuolar thiol protease, proaleurain, and eluted when the pH was lowered to 4. The protein was not retained on a control column prepared with the N-terminal sequence of a homologous, secreted thiol protease, endopeptidase B. The 80-kDa protein also accumulated in a membrane fraction that is less dense than clathrin-coated vesicles. In vitro studies demonstrated a binding constant of 37 nM between the =80-kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the "80-kDa protein, while a peptide carrying a single amino acid substitution known to abolish prosporamin vacuolar targeting had no measurable binding affinity for the protein. The binding protein is a glycoprotein with a transmembrane orientation in which the C terminus is exposed to the cytoplasm. The binding domain is located in the N-terminal luminal portion of the protein. These properties of the binding protein are consistent with the function of a receptor that would select proteins in the trans-Golgi for sorting to clathrin-coated vesicles and delivery to the vacuole.Vacuoles are acidic compartments occupying up to 80% of the volume of mature plant cells. In addition to functioning in the maintenance ofturgor and as a depository for such solutes as amino acids, sugars, organic acids, and mineral salts, the vacuoles also contain hydrolytic enzymes and, at certain stages of plant development, may serve as sites of accumulation of storage proteins (1). The accumulation of hydrolytic enzymes has been used as evidence to consider that the vacuole is a lytic compartment in plants analogous to the lysosome of the mammalian cell (2). Because of the presence of hydrolytic enzymes and the accumulation of reserve proteins, the vacuole/protein body is a useful system in which to study protein targeting in plants (3, 4). As in the mammalian lysosome system, soluble vacuolar proteins are synthesized in the endoplasmic reticulum and progress through the Golgi apparatus and clathrin-coated vesicles (CCVs) (5, 6) prior to accumulating in the vacuole. Although some soluble vacuolar proteins are glycosylated, N-linked oligosaccharides have no role in sorting glycoproteins to the vacuole (3, 4, 7). There is an accumulating body of information indicating that targeting of soluble proteins to the vacuole is mediated by determinants that reside in the polypeptide (3, 4, 7). To date, N-terminal (8-11) and C-terminal targeting determinant sequences (12, 13) have been identified; internal peptide sequences also appear to be in...

The processes by which soluble proteins are sorted from the secretory pathway to the vacuolar compartments in plant cells are poorly understood. In contrast to receptormediated sorting of lysosomal proteins in mammalian cells, where the sorting determinant is a Man-6-P residue added to Asn-linked oligosaccharides (Kornfeld, 1992), plant vacuolar sorting is determined by sequences within the polypeptides themselves (Bednarek and Raikhel, 1991;Matsuoka and Nakamura, 1991;Neuhaus et al., 1991;Saalbach et al., 1991;Holwerda et al., 1992). A similar strategy is used in yeast, where the tetrapeptide QRPL within the . '

The tonoplast was proposed as a default destination of membrane-bound proteins without specific targeting signals. To investigate the nature of this targeting, we created type I fusion proteins with green fluorescent protein followed by the transmembrane domain of the human lysosomal protein LAMP1. We varied the length of the transmembrane domain from 23 to either 20 or 17 amino acids by deletion within the hydrophobic domain. The resulting chimeras, called TM23, TM20, and TM17, were expressed either transiently or stably in tobacco. TM23 clearly accumulated in the plasmalemma, as confirmed by immunoelectron microscopy. In contrast, TM17 clearly was retained in the endoplasmic reticulum, and TM20 accumulated in small mobile structures. The nature of the TM20-labeled compartments was investigated by coexpression with a marker localized mainly in the Golgi apparatus, AtERD2, fused to a yellow fluorescent protein. The strict colocalization of both fluorescent proteins indicated that TM20 accumulated in the Golgi apparatus. To further test the default destination of type I membrane proteins, green fluorescent protein was fused to the 19-amino acid transmembrane domain of the plant vacuolar sorting receptor BP-80. The resulting chimera also accumulated in the Golgi instead of in post-Golgi compartments, where native BP-80 localized. Additionally, when the transmembrane domain of BP-80 was lengthened to 22 amino acids, the reporter escaped the Golgi and accumulated in the plasma membrane. Thus, the tonoplast apparently is not a favored default destination for type I membrane proteins in plants. Moreover, the target membrane where the chimera concentrates is not unique and depends at least in part on the length of the membrane-spanning domain. INTRODUCTIONThe sorting of integral proteins in plants is not well understood. Nevertheless, it is accepted that peptidic signals exposed in the cytosol are responsible for targeting to the correct subcellular location in plant cells. This signal-mediated sorting is opposed to a default transport that is believed to happen when no signal is present on a protein.Although the default destination within the secretory pathway for a soluble protein is secretion, the default membrane is unclear. The most informative results about the location where membrane proteins would accumulate by default were provided by a study of ␣ -TIP (Höfte and Chrispeels, 1992). In this experiment, the last 48 amino acids of ␣ -TIP, which contains the sixth transmembrane domain, were sufficient to target a reporter protein to the tonoplast. Because the deletion of the cytosolic C-terminal 15 amino acids from the ␣ -TIP sequence did not prevent the tonoplast accumulation of the truncated protein, the authors indirectly deduced a role for the sixth membrane-spanning domain. Either this ␣ -TIP transmembrane domain would be sufficient for vacuolar location or the tonoplast would be the default destination for membrane proteins.More recently, the same sixth transmembrane domain of ␣ -TIP was used in a chimeric c...

Rab GTPases are universal key regulators of intracellular secretory trafficking events. In particular, Rab 5 homologues have been implicated in endocytic events and in the vacuolar pathway. In this study, we investigate the location and function of a member of this family, AtRabF2b (Ara7) in tobacco (Nicotiana tabacum) leaf epidermal cells using a live cell imaging approach. Fluorescent-tagged AtRabF2b[wt] localized to the prevacuolar compartment and Golgi apparatus, as determined by coexpression studies with fluorescent markers for these compartments. Mutations that impair AtRabF2b function also alter the subcellular location of the GTPase. In addition, coexpression studies of the protein with the vacuole-targeted aleurain-green fluorescent protein (GFP) and rescue experiments with wild-type AtRabF2b indicate that the dominant-negative mutant of AtRabF2b causes the vacuolar marker to be secreted to the apoplast. Our results indicate a clear role of AtRabF2b in the vacuolar trafficking pathway.