Recently, an alternative route to the proteasomal protein-degradation pathway was discovered that specifically targets transmembrane proteins marked with a single ubiquitin to the endosomal multivesicular body (MVB) and,subsequently, to the vacuole (yeast) or lysosome (animals), where they are degraded by proteases. Vps23p/TSG101 is a key component of the ESCRT I-III machinery in yeast and animals that recognizes mono-ubiquitylated proteins and sorts them into the MVB. Here, we report that the Arabidopsis ELCH(ELC) gene encodes a Vps23p/TSG101 homolog, and that homologs of all known ESCRT I-III components are present in the Arabidopsis genome. As with its animal and yeast counterparts, ELC binds ubiquitin and localizes to endosomes. Gel-filtration experiments indicate that ELC is a component of a high-molecular-weight complex. Yeast two-hybrid and immunoprecipitation assays showed that ELC interacts with Arabidopsis homologs of the ESCRT I complex. The elc mutant shows multiple nuclei in various cell types,indicating a role in cytokinesis. Double-mutant analysis with kaktusshows that increased ploidy levels do not influence the cytokinesis effect of elc mutants, suggesting that ELC is only important during the first endoreduplication cycle. Double mutants with tubulin folding cofactor a mutants show a synergistic phenotype, suggesting that ELC regulates cytokinesis through the microtubule cytoskeleton.

SUMMARYThe vacuole is the most prominent organelle of plant cells. Despite its importance for many physiological and developmental aspects of plant life, little is known about its biogenesis and maintenance. Here we show that Arabidopsis plants expressing a dominant-negative version of the AAA (ATPase associated with various cellular activities) ATPase AtSKD1 (SUPPRESSOR OF K + TRANSPORT GROWTH DEFECT1) under the control of the trichome-specific GLABRA2 (GL2) promoter exhibit normal vacuolar development in early stages of trichome development. Shortly after its formation, however, the large central vacuole is fragmented and finally disappears completely. Secretion assays with amylase fused to the vacuolar sorting signal of Sporamin show that dominant-negative AtSKD1 inhibits vacuolar trafficking of the reporter that is instead secreted. In addition, trichomes expressing dominant-negative AtSKD1 frequently contain multiple nuclei. Our results suggest that AtSKD1 contributes to vacuolar protein trafficking and thereby to the maintenance of the large central vacuole of plant cells, and might play a role in cell-cycle regulation.

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo-and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

In yeast, endosomal sorting of monoubiquitylated transmembrane proteins is performed by a subset of the 19 "class E vacuolar protein sorting" proteins. The core machinery consists of 11 proteins that are organised in three complexes termed ESCRT I-III (endosomal sorting complex required for transport I-III) and is conserved in eukaryotic cells. While the pathway is well understood in yeast and animals, the plant ESCRT system is largely unexplored. At least one sequence homolog for each ESCRT component can be found in the Arabidopsis genome. Generally, sequence conservation between yeast/animals and the Arabidopsis proteins is low. To understand details about participating proteins and complex organization we have performed a systematic pairwise yeast two hybrid analysis of all Arabidopsis proteins showing homology to the ESCRT core machinery. Positive interactions were validated using bimolecular fluorescence complementation. In our experiments, most putative ESCRT components exhibited interactions with other ESCRT components that could be shown to occur on endosomes suggesting that despite their low homology to their yeast and animal counterparts they represent functional components of the plant ESCRT pathway.

Sorting of transmembrane proteins into the inner vesicles of multivesicular bodies for subsequent delivery to the vacuole/lysosome can be induced by attachment of a single ubiquitin or K63-linked ubiquitin chains to the cytosolic portion of the cargo in yeast and mammals. In plants, large efforts have been undertaken to elucidate the mechanisms of vacuolar trafficking of soluble proteins. Sorting of transmembrane proteins, by contrast, is still largely unexplored. As a proof of principle, that ubiquitin is involved in vacuolar sorting in plants we show that a translational fusion of a single ubiquitin to the Arabidopsis plasma membrane ATPase PMA-EGFP is sufficient to induce its endocytosis and sorting into the vacuolar lumen. Sorting of the artificial reporter is not dependent on ubiquitin chain formation, but involves ubiquitin's hydrophobic patch and can be inhibited by coexpression of a dominant-negative version of the ESCRT (endosomal sorting complex required for transport) related protein AtSKD1 (SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1). Our results suggest that ubiquitin can in principle act as vacuolar sorting signal in plants.