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. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation.

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. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation.

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. Here, we report on the proof-of-concept and early development stage of a collection of biofortified rice events with a high density of Fe and Zn in polished grains that have been pursued further to advance development for product release. In total, eight constructs were developed specifically expressing dicot ferritins and the rice nicotianamine synthase 2 (OsNAS2) gene under different combinations of promoters. A large-scale transformation of these constructs to Bangladesh and Philippines commercial indica cultivars and subsequent molecular screening and confined field evaluations resulted in the identification of a pool of ten events with Fe and Zn concentrations in polished grains of up to 11 µg g-1 and up to 37 µg g-1, respectively. The latter has the potential to reduce the prevalence of inadequate Zn intake for women of childbearing age in Bangladesh and in the Philippines by 30% and 50%, respectively, compared to the current prevalence. To our knowledge, this is the first potential biotechnology public-sector product that adopts the product cycle phase-gated approach, routinely applied in the private sector.

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. We use the degradation of Ypq1, a vacuole membrane lysine transporter, to demonstrate the protocol, which can be adapted for other organelle MPs and thus provide useful tools to study MP regulation in yeast and other model organisms. For complete details on the use and execution of this protocol, please refer to Arines et al. (2021) and Yang et al. (2020).

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. One model suggests that the ESCRT machinery is responsible for the sorting. In contrast, the ESCRT-independent intralumenal fragment (ILF) pathway proposes that the fragment generated by homotypic vacuole fusion is responsible for the sorting. Here, we applied a microfluidics-based imaging method to capture the complete degradation process in vivo. Combining live-cell imaging with a synchronized ubiquitination system, we demonstrated that ILF cargoes are not degraded through intralumenal fragments. Instead, ESCRTs function on the vacuole membrane to sort them into the lumen for degradation. We further discussed challenges in reconstituting vacuole membrane protein degradation.

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. We show evidence of an interaction between two transmembrane helices of Ypq1 (TM5 and TM7) and the single transmembrane helix of Ssh4. This interaction is regulated by the conserved PQ motif. Strikingly, recent structural studies of the PQ-loop family have suggested that TM5 and TM7 undergo major conformational changes during substrate transport, implying that transport-associated conformational changes may determine the selectivity. These findings thus provide critical information concerning the regulatory mechanism through which transmembrane domains can be specifically recognized in response to changing environmental conditions.

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. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.

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. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors.

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). Reports on engineering Fe content in rice have shown an increase up to 18 µg g(-1) in glasshouse settings; in contrast, under field conditions, 4 µg g(-1) was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 µg g(-1) Fe and 45.7 µg g(-1) Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

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.