Utilizing Plant Protein Secretion System for Pharmaceutical Protein Production.

Molecular farming, also known as plant molecular farming (PMF), is a technique that involves using plants and plant cells as bioreactors to produce recombinant proteins. This is a cost-effective and sustainable way of producing large quantities of proteins for various applications, including pharmaceuticals, vaccines, and industrial enzymes. An endogenous or exogenous signal peptide (SP) is flanked at the N-terminal for recombinant protein targeting and storage.

The plant unique ESCRT component FREE1 regulates autophagosome closure.

The energy sensor AMP-activated protein kinase (AMPK) can activate autophagy when cellular energy production becomes compromised. However, the degree to which nutrient sensing impinges on the autophagosome closure remains unknown. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1α1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation.

Ufmylation reconciles salt stress-induced unfolded protein responses via ER-phagy in .

In plants, the endomembrane system is tightly regulated in response to environmental stresses for maintaining cellular homeostasis. Autophagosomes, the double membrane organelles forming upon nutrient deprivation or stress induction, degrade bulky cytosolic materials for nutrient turnover. Though abiotic stresses have been reported to induce plant autophagy, few receptors or regulators for selective autophagy have been characterized for specific stresses.

In vitro reconstitution of COPII vesicles from Arabidopsis thaliana suspension-cultured cells.

Transport vesicles mediate protein traffic between endomembrane organelles in a highly selective and efficient manner. In vitro reconstitution systems have been widely used for studying mechanisms of vesicle formation, polar trafficking, and cargo specificity in mammals and yeast. However, this technique has not yet been applied to plants because of the large lytic vacuoles and rigid cell walls.

An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells.

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 pre-vacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here we developed and tested an in vivo expression system for the identification of VSR cargos which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture medium along with their corresponding cargo proteins.

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus.

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.

The rice RMR1 associates with a distinct prevacuolar compartment for the protein storage vacuole pathway.

Transport of vacuolar proteins from Golgi apparatus or trans-Golgi network (TGN) to vacuoles is a receptor-mediated process via an intermediate membrane-bound prevacuolar compartment (PVC) in plant cells. Both vacuolar sorting receptor (VSR) and receptor homology region-transmembrane domain-RING-H2 (RMR) proteins have been shown to function in transporting storage proteins to protein storage vacuole (PSV), but little is known about the nature of the PVC for the PSV pathway. Here, we use the rice RMR1 (OsRMR1) as a probe to study the PSV pathway in plants.

EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells.

The exocyst protein complex mediates vesicle fusion with the plasma membrane. By expressing an (X)FP-tagged Arabidopsis thaliana homolog of the exocyst protein Exo70 in suspension-cultured Arabidopsis and tobacco (Nicotiana tabacum) BY-2 cells, and using antibodies specific for Exo70, we detected a compartment, which we term EXPO (for exocyst positive organelles). Standard markers for the Golgi apparatus, the trans-Golgi network/early endosome, and the multivesicular body/late endosome in plants do not colocalize with EXPO.

Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies.

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination.

Dynamic response of prevacuolar compartments to brefeldin a in plant cells.

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells.

Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells.

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs). We have demonstrated recently that vacuolar sorting receptor (VSR) proteins are concentrated on PVCs. In this study, we generated transgenic Nicotiana tabacum (tobacco) BY-2 cell lines expressing two yellow fluorescent protein (YFP)-fusion reporters that mark PVC and Golgi organelles.

BP-80 and homologs are concentrated on post-Golgi, probable lytic prevacuolar compartments.

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between Golgi and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins.