Protocol for purifying and reconstituting a vacuole membrane transporter Ypq1 into proteoliposomes.

Studying the biochemical function of membrane transporters is important in understanding the biology of transporter-laden organelles such as lysosomes and vacuoles. We present a protocol for overexpressing, purifying, and reconstituting a vacuole membrane transporter Ypq1 into proteoliposomes and describe steps to measure transport activity using radioactive substrates. The protocols established here can be used to study other vacuolar or lysosomal membrane transporters.

Lysosomal membrane transporter purification and reconstitution for functional studies.

Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles.

Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation.

Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles.

Proof of concept and early development stage of market-oriented high iron and zinc rice expressing dicot ferritin and rice nicotianamine synthase genes.

Micronutrient deficiencies such as iron (Fe), zinc (Zn), and vitamin A, constitute a severe global public health phenomenon. Over half of preschool children and two-thirds of nonpregnant women of reproductive age worldwide have micronutrient deficiencies. Biofortification is a cost-effective strategy that comprises a meaningful and sustainable means of addressing this issue by delivering micronutrients through staple foods to populations with limited access to diverse diets and other nutritional interventions.

An optimized protocol to analyze membrane protein degradation in yeast using quantitative western blot and flow cytometry.

Membrane proteins (MPs) are essential in many cellular functions. To maintain proteostasis, MPs are downregulated via ubiquitination and degradation. Here, we describe an optimized protocol to analyze MP degradation using quantitative western blot and flow cytometry-based approaches.

ESCRT, not intralumenal fragments, sorts ubiquitinated vacuole membrane proteins for degradation.

The lysosome (or vacuole in fungi and plants) is an essential organelle for nutrient sensing and cellular homeostasis. In response to environmental stresses such as starvation, the yeast vacuole can adjust its membrane composition by selectively internalizing membrane proteins into the lumen for degradation. Regarding the selective internalization mechanism, two competing models have been proposed.

A selective transmembrane recognition mechanism by a membrane-anchored ubiquitin ligase adaptor.

While it is well-known that E3 ubiquitin ligases can selectively ubiquitinate membrane proteins in response to specific environmental cues, the underlying mechanisms for the selectivity are poorly understood. In particular, the role of transmembrane regions, if any, in target recognition remains an open question. Here, we describe how Ssh4, a yeast E3 ligase adaptor, recognizes the PQ-loop lysine transporter Ypq1 only after lysine starvation.

TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy.

Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the up-regulation of autophagy and vacuole proteins involved in recycling but also result in the down-regulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Down-regulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1.

Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system.

The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1.

Biofortified indica rice attains iron and zinc nutrition dietary targets in the field.

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 μg g(-1) iron (Fe) and 16 μg g(-1) zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 μg g(-1) Fe and 28 μg g(-1) Zn to reach approximately 30% of the estimated average requirement (EAR).

Molecular Analyses of Transgenic Plants.

One of the major challenges in plant molecular biology is to generate transgenic plants that express transgenes stably over generations. Here, we describe some routine methods to study transgene locus structure and to analyze transgene expression in plants: Southern hybridization using DIG chemiluminescent technology for characterization of transgenic locus, SYBR Green-based real-time RT-PCR to measure transgene transcript level, and protein immunoblot analysis to evaluate accumulation and stability of transgenic protein product in the target tissue.