Divergent functions of VTI12 and VTI11 in trafficking to storage and lytic vacuoles in Arabidopsis

The protein storage vacuole (PSV) is a plant-specific organelle that accumulates reserve proteins, one of the main agricultural products obtained from crops. Despite the importance of this process, the cellular machinery required for transport and accumulation of storage proteins remains largely unknown. Interfering with transport to PSVs has been shown to result in secretion of cargo. Therefore, secretion of a suitable marker could be used as an assay to identify mutants in this pathway. CLV3, a negative regulator of shoot stem cell proliferation, is an extracellular ligand that is rendered inactive when targeted to vacuoles. We devised an assay where trafficking mutants secrete engineered vacuolar CLV3 and show reduced meristems, a phenotype easily detected by visual inspection of plants. We tested this scheme in plants expressing VAC2, a fusion of CLV3 to the vacuolar sorting signal from the storage protein barley lectin. In this way, we determined that trafficking of VAC2 requires the SNARE VTI12 but not its close homologue, the conditionally redundant VTI11 protein. Furthermore, a vti12 mutant is specifically altered in transport of storage proteins, whereas a vti11 mutant is affected in transport of a lytic vacuole marker. These results demonstrate the specialization of VTI12 and VTI11 in mediating trafficking to storage and lytic vacuoles, respectively. Moreover, they validate the VAC2 secretion assay as a simple method to isolate genes that mediate trafficking to the PSV

The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

Subcellular trafficking is required for a multitude of functions in eukaryotic cells. It involves regulation of cargo sorting, vesicle formation, trafficking and fusion processes at multiple levels. Adaptor protein (AP) complexes are key regulators of cargo sorting into vesicles in yeast and mammals but their existence and function in plants have not been demonstrated. Here we report the identification of the protein-affected trafficking 4 (pat4) mutant defective in the putative d subunit of the AP-3 complex. pat4 and pat2, a mutant isolated from the same GFP imaging-based forward genetic screen that lacks a functional putative AP-3 ß, as well as dominant negative AP-3 µ transgenic lines display undistinguishable phenotypes characterized by largely normal morphology and development, but strong intracellular accumulation of membrane proteins in aberrant vacuolar structures. All mutants are defective in morphology and function of lytic and protein storage vacuoles (PSVs) but show normal sorting of reserve proteins to PSVs. Immunoprecipitation experiments and genetic studies revealed tight functional and physical associations of putative AP-3 ß and AP-3 d subunits. Furthermore, both proteins are closely linked with putative AP-3 µ and s subunits and several components of the clathrin and dynamin machineries. Taken together, these results demonstrate that AP complexes, similar to those in other eukaryotes, exist in plants, and that AP-3 plays a specific role in the regulation of biogenesis and function of vacuoles in plant cells

The Secretory System of Arabidopsis

Over the past few years, a vast amount of research has illuminated the workings of the secretory system of eukaryotic cells. The bulk of this work has been focused on the yeast Saccharomyces cerevisiae, or on mammalian cells. At a superficial level, plants are typical eukaryotes with respect to the operation of the secretory system; however, important differences emerge in the function and appearance of endomembrane organelles. In particular, the plant secretory system has specialized in several ways to support the synthesis of many components of the complex cell wall, and specialized kinds of vacuole have taken on a protein storage role—a role that is intended to support the growing seedling, but has been co-opted to support human life in the seeds of many crop plants. In the past, most research on the plant secretory system has been guided by results in mammalian or fungal systems but recently plants have begun to stand on their own as models for understanding complex trafficking events within the eukaryotic endomembrane system