Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H + -pyrophosphatase and the vacuolar H + -adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)-Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.

Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplastlocalized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANS-PORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the b-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast.

SUMMARY Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is not necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.

Uptake of carbohydrates across plasma membranes of animal cells is mediated mainly by Na + -driven transporters, such as the sodium-dependent glucose transporter(SGLT) (1) or the sodium-myo-inositol co-transporter (SMIT) (2). In contrast, animal monosaccharide transporters of the GLUT family (GLUT1 to GLUT12) are energy-independent (1). One protein of this family, however, GLUT13, acts as energy-dependent H + -inositol symporter (HMIT) (3). All 13 GLUT proteins are closely related to H + -dependent sugar transporters from bacteria and plants (4). Plants do not have SGLT-like or SMIT-like proteins. They rather catalyze the transport of inositol with several HMIT-like H + -symporters [three in Arabidopsis thaliana: AtINT1, AtINT2 and AtINT4 (5-7)] that localize either to the plasma membrane (AtINT2 and AtINT4) or to the tonoplast (AtINT1).Inositols have multiple and partly different metabolic functions in plants and animals, and in both kingdoms, inositol derivatives are central for cellular signaling (8-10). Altered cytoplasmic inositol concentrations might, therefore, affect the general metabolism as well as specific signaling pathways. In the brains of patients suffering from depression or bipolar disorders, increased inositol concentrations are thought to affect cellular signaling and behavior (11). As neurons cannot synthesize inositol (12,13), increased neuronal inositol concentrations might be explained by altered HMIT-catalyzed inositol uptake (13). Similarly, in plants, a defect in a single inositol transporter causes severe developmental defects (7).Obviously, cytoplasmic inositol concentrations are regulated. The rapid cycling between intracellular vesicles and the cell surface described for the HMIT protein in rat (Rattus norvegicus) neurons (13) demonstrates the tight control of inositol uptake. Moreover, since more than 50 years, treatments of mood disorders rely on drugs (Li + salts, valproic acid, etc.) known to reduce cellular inositol concentrations by inhibiting enzymes involved in the recycling of inositol (11).Inositol uptake might also be regulated by extracellular factors or changing environmental conditions. Interestingly, all animal and plant plasma-membrane H + -inositol symporters (but not the H + -inositol symporters from bacteria, fungi or plant endomembranes) carry a large extracellular loop domain between their predicted transmembrane helices IX and X (IX/X-loop). This domain contains eight highly conserved cysteines, four as CXXC motifs (CXXC, two cysteines separated by two other amino acids), but information on the function of this domain is lacking.Here, we present in-depth analyses of the IX/X-loop domains of plasma-membrane inositol transporters from plants and animals. Comparative studies performed with the human HMIT protein (hHMIT) and the A. thaliana INT2 protein (AtINT2) revealed that their IX/X-loops share sequence and structural similarity with plexin/semaphorin/ integrin (PSI) domains previously found in the extracellular regions of animal type I receptors including the ...

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