Molecular Characterization of Vacuolar Sorting Receptor-cargo Interaction in Arabidopsis

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the prevacuolar compartment (PVC) /multivesicular body (MVB). However, little is known about native VSR cargo proteins in plants. Here I developed and tested an in vivo expression system for the identification of VSR cargos which is based on the premise that the expressed Nterminus of VSRs will be secreted into the culture media along with their corresponding cargo proteins. Indeed, transgenic Arabidopsis culture cell lines expressing VSRNTs were shown to secrete truncated VSRs (BP80NT, AtVSR1NT and AtVSR4NT) with attached cargo molecules into the culture media. Putative cargo proteins were identified through mass spectrometry. Several identified cargo proteins were confirmed by their localization studies and interaction analysis with VSRs. The screening strategy described here should be applicable to all VSRs and will help identify and study cargo proteins for individual VSR proteins. This method should be useful for both cargo identification and protein--protein interaction in vivo.;The pH of intracellular compartments is essential for the viability of cells. ii Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, I have firstly generated two pH sensors by combining the improvedsolubility feature of solubility-modified green fluorescent protein (smGFP) with the pH-sensing capability of the pHluorins and codon optimized for expression in Arabidopsis. Plant-solubility-modified ecliptic pHluorin (PEpHluorin) gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and plantsolubility- modified ratiomatric pHluorin (PRpHluorin), a dual-excitation sensor, allowing for precise measurements. Compartment-specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Results show that the pH of cytosol and nucleus is similar (pH 7.3 and 7.2), while peroxisomes, mitochondrial matrix and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum (ER) to pH about 5.2 in the vacuole. Surprisingly, pH in the TGN and PVC is with pH 6.3 and 6.2 quite similar. The inhibition of vacuolar-type H+-ATPase (V-ATPase) with concanamycin A (ConcA) caused drastic increase of pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo respectively.