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

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

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

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

Artificial membranes for membrane protein purification, functionality and structure studies.

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method

Molecular interactions of the mitochondrial Tim12 translocase subunit

The small Tims are chaperones that facilitate insertion of hydrophobic precursors into the inner mitochondrial membrane. We purified Tim12 and found it forms dimers that bind to Tim9. In this interaction, Tim12 undergoes structural changes that may be important for transport of its substrates in the mitochondrial carrier import pathway.</p

Routes to and from the plasma membrane:Bulk flow versus signal mediated endocytosis

Transport of proteins via the secretory pathway is controlled by a combination of signal dependent cargo selection as well as unspecific bulk flow of membranes and aqueous lumen. Using the plant vacuolar sorting receptor as model for membrane spanning proteins, we have distinguished bulk flow from signal mediated protein targeting in biosynthetic and endocytic transport routes and investigated the influence of transmembrane domain length. More specifically, long transmembrane domains seem to prevent ER retention, either by stimulating export or preventing recycling from post ER compartments. Long transmembrane domains also seem to prevent endocytic bulk flow from the plasma membrane, but the presence of specific endocytosis signals overrules this in a dominant manner

Routes to and from the plasma membrane: bulk flow versus signal mediated endocytosis

Transport of proteins via the secretory pathway is controlled by a combination of signal dependent cargo selection as well as unspecific bulk flow of membranes and aqueous lumen. Using the plant vacuolar sorting receptor as model for membrane spanning proteins, we have distinguished bulk flow from signal mediated protein targeting in biosynthetic and endocytic transport routes and investigated the influence of transmembrane domain length. More specifically, long transmembrane domains seem to prevent ER retention, either by stimulating export or preventing recycling from post ER compartments. Long transmembrane domains also seem to prevent endocytic bulk flow from the plasma membrane, but the presence of specific endocytosis signals overrules this in a dominant manner

Peroxisomal transport systems: roles in signaling and metabolism

Peroxisomes perform a range of different functions, including β-oxidation of fatty acids and synthesis and degradation of bioactive molecules. A notable feature of peroxisomes is their role in metabolic pathways which are shared between several subcellular compartments, including mitochondria, chloroplasts and cytosol. Transport across the peroxisomal membrane is therefore central to the co-ordination of metabolism. Although transport processes are required for import of substrates and cofactors, export of intermediates and products and the operation of redox shuttles, relatively few peroxisomal transporters have been identified to date. This chapter reviews the current evidence for and against different peroxisomal transport processes