Membrane Proteins From A to Z Lysosomal and vacuolar sorting: not so different after all!

Abstract Soluble hydrolases represent the main proteins of lysosomes and vacuoles and are essential to sustain the lytic properties of these organelles typical for the eukaryotic organisms. The sorting of these proteins from ER residents and secreted proteins is controlled by highly specific receptors to avoid mislocalization and subsequent cellular damage. After binding their soluble cargo in the early stage of the secretory pathway, receptors rely on their own sorting signals to reach their target organelles for ligand delivery, and to recycle back for a new round of cargo recognition. Although signals in cargo and receptor molecules have been studied in human, yeast and plant model systems, common denominators and specific examples of diversification have not been systematically explored. This review aims to fill this niche by comparing the structure and the function of lysosomal/vacuolar sorting receptors (VSRs) from these three organisms

Unconventional Secretion of Plant Extracellular Vesicles and Their Benefits to Human Health: A Mini Review.

Mechanisms devoted to the secretion of proteins via extracellular vesicles (EVs) have been found in mammals, yeasts, and plants. Since they transport a number of leader-less proteins to the plasma membrane or the extracellular space, EVs are considered part of Unconventional protein secretion (UPS) routes. UPS involving EVs are a relatively new field in plants. Aside from their role in plant physiology and immunity, plant extracts containing EVs have also been shown to be beneficial for human health. Therefore, exploring the use of plant EVs in biomedicine and their potential as drug delivery tools is an exciting avenue. Here we give a summary of the state of knowledge on plant EVs, their crosstalk with mammalian systems and potential research routes that could lead to practical applications in therapeutic drug delivery

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.Peer reviewe

The NBDs that wouldn't die: A cautionary tale of the use of isolated nucleotide binding domains of ABC transporters

COMATOSE (CTS), the plant homologue of Adrenoleukodystrophy protein, is a full length ABC transporter localised in peroxisomes. In a recent article, we reported that the two nucleotide binding domains of CTS are not functionally equivalent in vivo. Mutations in conserved residues in the Walker A (K487A) and B (D606N) motifs of NBD1 resulted in a null phenotype, whereas identical mutations in the equivalent residues in NBD2 (K1136A and D1276N) had no detectable effect.1 In order to study the effect of these mutations on the ATPase activity of the nucleotide binding domains, we cloned and expressed the isolated NBDs as maltose binding protein (MBP) fusion proteins. We show that ATPase activity is associated with the isolated MBP-NBDs. However, mutations of amino acids located in conserved motifs did not result in striking reduction in activity despite well characterized roles in ATP binding and hydrolysis. We urge caution in the interpretation of results obtained from the study of isolated NBD fusions and their extrapolation to the mechanism of ATP hydrolysis in ABC transporter proteins

Trafficking routes to the plant vacuole: connecting alternative and classical pathways

Due to the numerous roles plant vacuoles play in cell homeostasis, detoxification, and protein storage, the trafficking pathways to this organelle have been extensively studied. Recent evidence, however, suggests that our vision of transport to the vacuole is not as simple as previously imagined. Alternative routes have been identified and are being characterized. Intricate interconnections between routes seem to occur in various cases, complicating the interpretation of data. In this review, we aim to summarize the published evidence and link the emerging data with previous findings. We discuss the current state of information on alternative and classical trafficking routes to the plant vacuole

Subcellular localization and trafficking of phytolongins (non-SNARE longins) in the plant secretory pathway.

SNARE proteins are central elements of the machinery involved in membrane fusion of eukaryotic cells. In animals and plants, SNAREs have diversified to sustain a variety of specific functions. In animals, R-SNARE proteins called brevins have diversified; in contrast, in plants, the R-SNARE proteins named longins have diversified. Recently, a new subfamily of four longins named 'phytolongins' (Phyl) was discovered. One intriguing aspect of Phyl proteins is the lack of the typical SNARE motif, which is replaced by another domain termed the 'Phyl domain'. Phytolongins have a rather ubiquitous tissue expression in Arabidopsis but still await intracellular characterization. In this study, we found that the four phytolongins are distributed along the secretory pathway. While Phyl2.1 and Phyl2.2 are strictly located at the endoplasmic reticulum network, Phyl1.2 associates with the Golgi bodies, and Phyl1.1 locates mainly at the plasma membrane and partially in the Golgi bodies and post-Golgi compartments. Our results show that export of Phyl1.1 from the endoplasmic reticulum depends on the GTPase Sar1, the Sar1 guanine nucleotide exchange factor Sec12, and the SNAREs Sec22 and Memb11. In addition, we have identified the Y48F49 motif as being critical for the exit of Phyl1.1 from the endoplasmic reticulum. Our results provide the first characterization of the subcellular localization of the phytolongins, and we discuss their potential role in regulating the secretory pathway

Exploiting the Opportunity to Use Plant-Derived Nanoparticles as Delivery Vehicles.

The scientific community has become increasingly interested in plant-derived nanoparticles (PDNPs) over the past ten years. Given that they possess all the benefits of a drug carrier, including non-toxicity, low immunogenicity, and a lipid bilayer that protects its content, PDNPs are a viable model for the design of innovative delivery systems. In this review, a summary of the prerequisites for mammalian extracellular vesicles to serve as delivery vehicles will be given. After that, we will concentrate on providing a thorough overview of the studies investigating the interactions of plant-derived nanoparticles with mammalian systems as well as the loading strategies for encapsulating therapeutic molecules. Finally, the existing challenges in establishing PDNPs as reliable biological delivery systems will be emphasized

Salt Stress Causes Peroxisome Proliferation, but Inducing Peroxisome Proliferation Does Not Improve NaCl Tolerance in Arabidopsis thaliana

The PEX11 family of peroxisome membrane proteins have been shown to be involved in regulation of peroxisome size and number in plant, animals, and yeast cells. We and others have previously suggested that peroxisome proliferation as a result of abiotic stress may be important in plant stress responses, and recently it was reported that several rice PEX11 genes were up regulated in response to abiotic stress. We sought to test the hypothesis that promoting peroxisome proliferation in Arabidopsis thaliana by over expression of one PEX11 family member, PEX11e, would give increased resistance to salt stress. We could demonstrate up regulation of PEX11e by salt stress and increased peroxisome number by both PEX11e over expression and salt stress, however our experiments failed to find a correlation between PEX11e over expression and increased peroxisome metabolic activity or resistance to salt stress. This suggests that although peroxisome proliferation may be a consequence of salt stress, it does not affect the ability of Arabidopsis plants to tolerate saline conditions

The NBDs that wouldn't die. A cautionary tale in the use of isolated nucleotide binding domains of ABC transporters

COMATOSE (CTS), the plant homologue of Adrenoleukodystrophy protein, is a full length ABC transporter localised in peroxisomes. In a recent article, we reported that the two nucleotide binding domains of CTS are not functionally equivalent in vivo. Mutations in conserved residues in the Walker A (K487A) and B (D606N) motifs of NBD1 resulted in a null phenotype, whereas identical mutations in the equivalent residues in NBD2 (K1136A and D1276N) had no detectable effect.1 In order to study the effect of these mutations on the ATPase activity of the nucleotide binding domains, we cloned and expressed the isolated NBDs as maltose binding protein (MBP) fusion proteins. We show that ATPase activity is associated with the isolated MBP-NBDs. However, mutations of amino acids located in conserved motifs did not result in striking reduction in activity despite well characterized roles in ATP binding and hydrolysis. We urge caution in the interpretation of results obtained from the study of isolated NBD fusions and their extrapolation to the mechanism of ATP hydrolysis in ABC transporter proteins

Lysosomal and vacuolar sorting: not so different after all!

Soluble hydrolases represent the main proteins of lysosomes and vacuoles and are essential to sustain the lytic properties of these organelles typical for the eukaryotic organisms. The sorting of these proteins from ER residents and secreted proteins is controlled by highly specific receptors to avoid mislocalization and subsequent cellular damage. After binding their soluble cargo in the early stage of the secretory pathway, receptors rely on their own sorting signals to reach their target organelles for ligand delivery, and to recycle back for a new round of cargo recognition. Although signals in cargo and receptor molecules have been studied in human, yeast and plant model systems, common denominators and specific examples of diversification have not been systematically explored. This review aims to fill this niche by comparing the structure and the function of lysosomal/vacuolar sorting receptors (VSRs) from these three organisms