A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition.

Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ-aminobutyrate (GABA) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans-stimulated in counterexchange mode by GABA, Glu and Asp. We identified SlCAT9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast-enriched membrane fraction. Transient expression of a SlCAT9-YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of SlCAT9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild-type (WT) only when assayed in counterexchange mode with Glu, Asp, or GABA. Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that SlCAT9 is a tonoplast Glu/Asp/GABA exchanger that strongly influences the accumulation of these amino acids during fruit development.

Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp.

This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development.

The role of CDC48 in the retro-translocation of non-ubiquitinated toxin substrates in plant cells.

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterize the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that although RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesized RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated.

Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting.

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting. Consequently, overexpression of BP80 partially restores vacuolar cargo transport. To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP). The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles. Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80. This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment. Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted. We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive.