Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins.     

A workflow for peptide-based proteomics in a poorly sequenced plant: a case study on the plasma membrane proteome of banana

Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified.     

Aquaporins are multifunctional water and solute transporters highly divergent in living organisms

Aquaporins (AQPs) are ubiquitous membrane proteins whose identification, pioneered by Peter Agre's team in the early nineties, provided a molecular basis for transmembrane water transport, which was previously thought to occur only by free diffusion. AQPs are members of the Major Intrinsic Protein (MIP) family and often referred to as water channels. In mammals and plants they are present in almost all organs and tissues and their function is mostly associated to water molecule movement. However, recent studies have pointed out a wider range of substrates for these proteins as well as complex regulation levels and pathways. Although their relative abundance in plants and mammals makes it difficult to investigate the role of a particular AQP, the use of knock-out and mutagenesis techniques is now bringing important clues regarding the direct implication of specific AQPs in animal pathologies or plant deficiencies. The present paper gives an overview about AQP structure, function and regulation in a broad range of living organisms. Emphasis will be given on plant AQPs where the high number and diversity of these transport proteins, together with some emerging aspects of their functionalities, make them behave more like multifunctional, highly adapted channels rather than simple water pores.     

Mammalian plasma membrane proteomics

Plasma membrane proteins serve essential functions for cells, interacting with both cellular and extracellular components, structures and signaling molecules. Additionally, plasma membrane proteins comprise more than two-thirds of the known protein targets for existing drugs. Consequently, defining membrane proteomes is crucial to understanding the role of plasma membranes in fundamental biological processes and for finding new targets for action in drug development. MS-based identification methods combined with chromatographic and traditional cell-biology techniques are powerful tools for proteomic mapping of proteins from organelles. However, the separation and identification of plasma membrane proteins remains a challenge for proteomic technology because of their hydrophobicity and microheterogeneity. Creative approaches to solve these problems and potential pitfalls will be discussed. Finally, a representative overview of the impressive achievements in this field will also be given.     

包封膜蛋白的仿生细胞膜

本文概述了脂质囊泡的组成成分和制作方法以及用于膜蛋白方面研究的相关技术,包括膜蛋白整合到囊泡的方法、复合体系的表征等。脂质囊泡可以为膜蛋白提供类似体内的环境,包括疏水区和内外亲水环境,因其组分单一,可以方便地进行结构、功能、信号转导等方面的研究,因此可以模拟细胞膜作为研究膜蛋白的有力工具,目前大多是以脂质体形态作为仿生囊泡体系进行这方面研究。     

Transport of electrolytes in muscle

The muscle fiber stands alongside the red blood cell and the giant axon as one of the three classical cell types that have had major application in investigating ion transport processes in cell membranes. Of these three cell types, the muscle fiber was the first to provide definite evidence for a sodium pump. The ability of the sodium pump to produce an electrical potential difference across the cell membrane was also first demonstrated in muscle fibers. This important property of the sodium pump is now known to have physiological significance in many other types of cells. In this review, electrolyte transport investigations in skeletal muscle are traced from their inception to the current state of the field. Applications of major research techniques are discussed and key results are summarized. An overview of electrolyte transport in muscle, this article emphasizes relationships between the muscle fiber membrane potential and ionic transport processes.     

PROCEED: A proteomic method for analysing plasma membrane proteins in living mammalian cells.

Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates. Exploring the realm of these proteins has proved to be an intricate task, mainly due to their hydrophobic nature and low abundance. Furthermore, the level of purity achieved by classical methods of purification and cell fractionation is insufficient. These restrictions pose major limitations for gel electrophoresis or chromatography-based separation techniques as the preferred methodologies for high-throughput analysis. Mass spectrometry has alleviated most of the difficulties in the identification of proteins in general; however, the Achilles' heel is still the isolation and separation of membrane proteins. In order to circumvent these limitations, a high-throughput platform has been devised, whereby proteases are applied to whole intact living cells. The resulting peptide fragments are then analysed by liquid chromatology followed by tandem MS (LC-MS/MS) technology to provide a detailed profile of proteins exposed on the surface of the plasma membrane. This kind of protein trimming offers the advantages that no prior manipulation or fractionation of the cell is required, contaminating proteins are remarkably reduced and the procedure is adequate for high-throughput purposes. This method, referred to as PROCEED (PROteome of Cell Exposed Extracellular Domains) is compatible with isotope labelling techniques which facilitate comparative protein expression studies. The methodology is extendable to all cell types including yeast and bacteria. Finally, the advantages and the limitations of PROCEED are discussed in view of other current technologies.     

The plasma membrane transport systems and adaptation to salinity

Salt stress represents one of the environmental challenges that drastically affect plant growth and yield. Evidence suggests that glycophytes and halophytes have a salt tolerance mechanisms working at the cellular level, and the plasma membrane (PM) is believed to be one facet of the cellular mechanisms. The responses of the PM transport proteins to salinity in contrasting species/cultivars were discussed. The review provides a comprehensive overview of the recent advances describing the crucial roles that the PM transport systems have in plant adaptation to salt. Several lines of evidence were presented to demonstrate the correlation between the PM transport proteins and adaptation of plants to high salinity. How alterations in these transport systems of the PM allow plants to cope with the salt stress was also addressed. Although inconsistencies exist in some of the information related to the responses of the PM transport proteins to salinity in different species/cultivars, their key roles in adaptation of plants to high salinity is obvious and evident, and cannot be precluded. Despite the promising results, detailed investigations at the cellular/molecular level are needed in some issues of the PM transport systems in response to salinity to further evaluate their implication in salt tolerance.     

Plasma Membrane Protein Ubiquitylation and Degradation as Determinants of Positional Growth in Plants

Being sessile organisms, plants evolved an unparalleled plasticity in their post-embryonic development, allowing them to adapt and fine-tune their vital parameters to an ever-changing environment. Crosstalk between plants and their environment requires tight regulation of information exchange at the plasma membrane (PM). Plasma membrane proteins mediate such communication, by sensing variations in nutrient availability, external cues as well as by controlled solute transport across the membrane border. Localization and steady-state levels are essential for PM protein function and ongoing research identified cis- and trans-acting determinants, involved in control of plant PM protein localization and turnover. In this overview, we summarize recent progress in our understanding of plant PM protein sorting and degradation via ubiquitylation, a post-translational and reversible modification of proteins. We highlight characterized components of the machinery involved in sorting of ubiquitylated PM proteins and discuss consequences of protein ubiquitylation on fate of selected PM proteins. Specifically, we focus on the role of ubiquitylation and PM protein degradation in the regulation of polar auxin transport (PAT). We combine this regulatory circuit with further aspects of PM protein sorting control, to address the interplay of events that might control PAT and polarized growth in higher plants.     

Structural and functional characteristics of plant proteinase inhibitor-II (PI-II) family

Plant proteinase inhibitor-II (PI-II) proteins are one of the promising defensive proteins that helped the plants to resist against different kinds of unfavorable conditions. Different roles for PI-II have been suggested such as regulation of endogenous proteases, modulation of plant growth and developmental processes and mediating stress responses. The basic knowledge on genetic and molecular diversity of these proteins has provided significant insight into their gene structure and evolutionary relationships in various members of this family. Phylogenetic comparisons of these family genes in different plants suggested that the high rate of retention of gene duplication and inhibitory domain multiplication may have resulted in the expansion and functional diversification of these proteins. Currently, a large number of transgenic plants expressing PI-II genes are being developed for enhancing the defensive capabilities against insects, bacteria and pathogenic fungi. Much emphasis is yet to be given to exploit this ever expanding repertoire of genes for improving abiotic stress resistance in transgenic crops. This review presents an overview about the current knowledge on PI-II family genes, their multifunctional role in plant defense and physiology with their potential applications in biotechnology.     

Transport of electrolytes in muscle

Summary The muscle fiber stands alongside the red blood cell and the giant axon as one of the three classical cell types that have had major application in investigating ion transport processes in cell membranes. Of these three cell types, the muscle fiber was the first to provide definite evidence for a sodium pump. The ability of the sodium pump to produce an electrical potential difference across the cell membrane was also first demonstrated in muscle fibers. This important property of the sodium pump is now known to have physiological significance in many other types of cells.In this review, electrolyte transport investigations in skeletal muscle are traced from their inception to the current state of the field. Applications of major research techniques are discussed and key results are summarized. An overview of electrolyte transport in muscle, this article emphasizes relationships between the muscle fiber membrane potential and ionic transport processes.     

Energetics of membrane protein folding and stability

The critical role of membrane proteins in a myriad of biological and physiological functions has spawned numerous investigations over the past several decades with the long-term goal of identifying the molecular origins and energetic forces that stabilize these proteins within the membrane. Parallel structural and thermodynamics studies on several systems have provided significant insight regarding the driving forces governing folding, assembly, insertion, and translocation of membrane proteins. The present review surveys families of membrane-associated proteins including α-helical and β-barrel structures, viral surface receptors, and pore-forming toxins, citing representative proteins within each of these classes for further scrutiny in terms of structure-function relationships and global conformational stability. This overview presents seminal findings from pioneering studies on the energetics of membrane protein folding and stability to modern techniques that are exploiting the use of molecular genetics and single molecule studies. An overall consensus regarding the molecular origins of membrane protein stability is that a number of intrinsic properties resemble features of soluble proteins, yet there are distinct energetic differences arising from specific intra- and intermolecular interactions within the membrane. The combined efforts from structural, energetics, and dynamics approaches offer unique insights and improve our fundamental understanding of the driving forces dictating membrane protein folding and stability.     

Shedding light on the puzzle of drug-membrane interactions: Experimental techniques and molecular dynamics simulations

Lipid membranes work as barriers, which leads to inevitable drug-membrane interactions in vivo. These interactions affect the pharmacokinetic properties of drugs, such as their diffusion, transport, distribution, and accumulation inside the membrane. Furthermore, these interactions also affect their pharmacodynamic properties with respect to both therapeutic and toxic effects. Experimental membrane models have been used to perform in vitro assessment of the effects of drugs on the biophysical properties of membranes by employing different experimental techniques. In in silico studies, molecular dynamics simulations have been used to provide new insights at an atomistic level, which enables the study of properties that are difficult or even impossible to measure experimentally. Each model and technique has its advantages and disadvantages. Hence, combining different models and techniques is necessary for a more reliable study. In this review, the theoretical backgrounds of these (in vitro and in silico) approaches are presented, followed by a discussion of the pharmacokinetic and pharmacodynamic properties of drugs that are related to their interactions with membranes. All approaches are discussed in parallel to present for a better connection between experimental and simulation studies. Finally, an overview of the molecular dynamics simulation studies used for drug-membrane interactions is provided.     

泛素介导的植物膜蛋白转运及其研究方法

泛素(Ub)是一类小分子多肽, 可通过赖氨酸残基与靶蛋白结合, 进而决定靶蛋白的去向。泛素分子对靶蛋白进行特异性修饰的过程称为泛素化。相较于动物和酵母细胞, 植物细胞中泛素介导的蛋白动态循环, 尤其是膜蛋白胞吞动态循环研究相对滞后。随着生物化学以及显微技术的发展, 人们对泛素介导的植物细胞膜蛋白转运有了新的认识。该文阐述了泛素及类泛素在蛋白转运中的作用, 总结了泛素化(ubiquitylation)调控膜蛋白转运的分子生物学机制和常用的研究方法, 并对今后该领域的研究进行了展望。     

Polar delivery in plants; commonalities and differences to animal epithelial cells

Although plant and animal cells use a similar core mechanism to deliver proteins to the plasma membrane, their different lifestyle, body organization and specific cell structures resulted in the acquisition of regulatory mechanisms that vary in the two kingdoms. In particular, cell polarity regulators do not seem to be conserved, because genes encoding key components are absent in plant genomes. In plants, the broad knowledge on polarity derives from the study of auxin transporters, the PIN-FORMED proteins, in the model plant Arabidopsis thaliana. In animals, much information is provided from the study of polarity in epithelial cells that exhibit basolateral and luminal apical polarities, separated by tight junctions. In this review, we summarize the similarities and differences of the polarization mechanisms between plants and animals and survey the main genetic approaches that have been used to characterize new genes involved in polarity establishment in plants, including the frequently used forward and reverse genetics screens as well as a novel chemical genetics approach that is expected to overcome the limitation of classical genetics methods.     

Proteome imaging: a closer look at life's organization

Imaging the proteome is a term that is used in many different contexts. The term implies that the entire cohort of proteins and their modifications are visualized. This unfortunately is not the case. In this mini-review, a concise overview is provided on different imaging technologies that are currently used to investigate the structure, function and dynamics of proteins and their organization. These techniques have been selected for review based on the unique insights they provide in subsets of the proteome. These techniques have been illustrated with practical examples of their merits. Mass spectrometry-based imaging technologies are playing a key role in proteome research and have been reviewed in more detail. They hold the promise of detailed molecular insight in the spatial organization of living system.     

Protein S-acylation in plants (Review)

Membrane resident proteins are a common feature of biology yet many of these proteins are not integral to the membrane. These peripheral membrane proteins are often bound to the membrane by the addition of fatty acyl chains to the protein. This modification, known as S-acylation or palmitoylation, promotes very strong membrane association but is also reversible allowing for a high degree of control over membrane association. Many S-acylated proteins are resident in sterol, sphingolipid and saturated-lipid enriched microdomains indicating an important role for S-acylation in protein partitioning within membranes. This review summarises the current knowledge of S-acylation in plants. S-acylated proteins play a wide variety of roles in plants and affect Ca²⁺ signalling, K⁺ movement, stress signalling, small and heterotrimeric G-protein membrane association and partitioning, tubulin function as well as pathogenesis. Although the study of S-acylation is in its infancy in plants this review illustrates that S-acylation is extremely important for plant function and that there are many unexplored aspects of S-acylation in plants. A full summary of the techniques and methods available to study S-acylation in plants is also presented.     

Comparative biology of telomeres: Where plants stand

Telomeres are essential structures at the ends of eukaryotic chromosomes. Work on their structure and function began almost 70 years ago in plants and flies, continued through the Nobel Prize winning work on yeast and ciliates, and goes on today in many model and non-model organisms. The basic molecular mechanisms of telomeres are highly conserved throughout evolution, and our current understanding of how telomeres function is a conglomeration of insights gained from many different species. This review will compare the current knowledge of telomeres in plants with other organisms, with special focus on the functional length of telomeric DNA, the search for TRF homologs, the family of POT1 proteins, and the recent discovery of members of the CST complex.     

The membrane proteome of Halobacterium salinarum

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha-helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2-DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2-DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.