Mechanism for the selective conjugation of ubiquitin to phytochrome

The goal of this project is to understand at the molecular level how phytochrome functions and how intracellular proteins are degraded. Phytochrome is marked for degradation by covalent attachment of ubiquitin. Ubiquitin-phytochrome conjugates (UbP) were characterized with respect to formation kinetics, subcellular localization and site of ubiquitin attachment. UbP appears to be a general phenomenon during phytochrome degradation in a variety of species. UbP was isolated from oat seedlings and characterized. Residues 747-830 of phytochrome have been identified as a possible attachment site for ubiquitin. By placing the gene for etiolated phytochrome in tobacco we have created a transgenic system for over expressing phytochrome. The effects of this over expression are described, and it appears that tobacco degrades this foreign protein through formation of UbP. We have created a series of site-directed mutants of the oat phytochrome gene, and are in the process of characterizing them to determine sequence requirements for ubiquination. 8 refs., 1 fig. (MHB

AUTOPHAGY-RELATED14 and Its Associated Phosphatidylinositol 3-Kinase Complex Promote Autophagy in Arabidopsis

Phosphatidylinositol 3-phosphate (PI3P) is an essential membrane signature for both autophagy and endosomal sorting that is synthesized in plants by the class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of the VPS34 kinase, together with ATG6, VPS15, and either VPS38 or ATG14 as the fourth subunit. Although Arabidopsis (Arabidopsis thaliana) plants missing the three core subunits are infertile, vps38 mutants are viable but have aberrant leaf, root, and seed development, Suc sensing, and endosomal trafficking, suggesting that VPS38 and ATG14 are nonredundant. Here, we evaluated the role of ATG14 through a collection of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and T-DNA insertion mutants disrupting the two Arabidopsis paralogs. atg14a atg14b double mutants were relatively normal phenotypically but displayed pronounced autophagy defects, including reduced accumulation of autophagic bodies and cargo delivery during nutrient stress. Unexpectedly, homozygous atg14a atg14b vps38 triple mutants were viable but showed severely compromised rosette development and reduced fecundity, pollen germination, and autophagy, consistent with a need for both ATG14 and VPS38 to fully actuate PI3P biology. However, the triple mutants still accumulated PI3P, but they were hypersensitive to the PI3K inhibitor wortmannin, indicating that the ATG14/VPS38 component is not essential for PI3P synthesis. Collectively, the ATG14/VPS38 mutant collection now permits the study of plants altered in specific aspects of PI3P biology

Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90 and ROF1

Plant science