Membrane-mediated interactions – a physico-chemical basis for protein sorting

Abstract

Sorting of membrane proteins in eukaryotic cells is a complex yet vital task that involves several 10,000 molecular players. Sorting takes place not only along the early secretory pathway, i.e., between the endoplasmic reticulum and the Golgi apparatus, but also between other organelles, including exchange with the cell's plasma membrane. Traditionally, specific binary interactions between proteins have been made responsible for most of the protein sorting. A more active role of lipids, however, became visible in recent years. Not only do lipids in complex membranes show domain formation that may support/suppress sorting events, but also collective, membrane-mediated interactions have emerged as a robust physico-chemical mechanism to drive protein sorting. Here, we will review recent insights into these aspects.     

Membrane-mediated interactions--a physico-chemical basis for protein sorting

Sorting of membrane proteins in eukaryotic cells is a complex yet vital task that involves several 10,000 molecular players. Sorting takes place not only along the early secretory pathway, i.e., between the endoplasmic reticulum and the Golgi apparatus, but also between other organelles, including exchange with the cell's plasma membrane. Traditionally, specific binary interactions between proteins have been made responsible for most of the protein sorting. A more active role of lipids, however, became visible in recent years. Not only do lipids in complex membranes show domain formation that may support/suppress sorting events, but also collective, membrane-mediated interactions have emerged as a robust physico-chemical mechanism to drive protein sorting. Here, we will review recent insights into these aspects.     

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.     

Electrostatic interactions in the denatured state ensemble: their effect upon protein folding and protein stability

It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well as hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol^-1 to the stability of the DSE.     

Mechanisms and concepts paving the way towards a complete transport cycle of plant vacuolar sorting receptors

Delivery of proteins to the lytic vacuole in plants is a complex cascade of selective interactions that specifically excludes residents of the endoplasmic reticulum and secreted proteins. Vacuolar transport must be highly efficient to avoid mistargeting of hydrolytic enzymes to locations where they could be harmful. While plant vacuolar sorting signals have been well described for two decades, it is only during the last 5 years that a critical mass of data was gathered that begins to reveal how vacuolar sorting receptors (VSRs) may complete a full transport cycle. Yet, the field is far from reaching a consensus regarding the organelles that could be involved in vacuolar sorting, their potential biogenesis, and the ultimate recycling of membranes and protein machinery that maintain this pathway. This review will highlight the important landmarks in our understanding of VSR function and compare recent transport models that have been proposed so that an emerging picture of plant vacuolar sorting mechanisms can be drawn.     

Force spectroscopy studies on protein–ligand interactions: A single protein mechanics perspective

Protein–ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein–ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein–ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein–ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings.     

Selective sorting of cargo proteins into bacterial membrane vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.     

High-performance affinity chromatography: a powerful tool for studying serum protein binding

High-performance affinity chromatography (HPAC) is a method in which a biologically-related ligand is used as a stationary phase in an HPLC system. This approach is a powerful means for selectively isolating or quantitating agents in complex samples, but it can also be employed to study the interactions of biological systems. In recent years there have been numerous reports in which HPAC has been used to examine the interactions of drugs, hormones and other substances with serum proteins. This review discusses how HPAC has been used in such work. Particular attention is given to the techniques of zonal elution and frontal analysis. Various applications are provided for these techniques, along with a list of factors that need to be considered in their optimization and use. New approaches based on band-broadening studies and rapid immunoextraction are also discussed.     

Protein targeting to dense-core secretory granules

Regulated secretory proteins are stored within specialized vesicles known as secretory granules. It is not known how proteins are sorted into these organelles. Regulated proteins may possess targeting signals which interact with specific sorting receptors in the lumen of the trans-Golgi network (TGN) prior to their aggregation to form the characteristic dense-core of the granule. Alternatively, sorting may occur as the result of specific aggregation of regulated proteins in the TGN. Aggregates may be directed to secretory granules by interaction of a targeting signal on the surface with a sorting receptor. Novel targeting signals which confer on regulated proteins a tendency to aggregate under certain conditions, and in so doing cause them to be incorporated into secretory granules, have been implicated. Specific targeting signals may also play a role in directing membrane proteins to secretory granules.     

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic “leucine collar” that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate.     

Membrane trafficking pathways in Alzheimer's disease

Membrane proteins are constantly being trafficked in cells and the relevant proteins in Alzheimer's disease (AD), such as the amyloid precursor protein (APP) and its processing enzymes, are not exempted from that. Molecular cell biologists have been endeavoring to ascertain a roadmap for APP processing and trafficking in various cell types including neurons. This has led to the identification of numerous regulatory sorting mechanisms, protein-protein interactions and lipidic microenvironments that largely define how and where the substrate APP meets its processing enzymes. However, the cell biology of tau, and the formation of neurofibrillary tangles, has long been regarded as a separate field. Nonetheless, recent progress is bringing both worlds together in a new paradigm on how Aβ toxicity and tau are physiologically connected. Here, we discuss an update of our current appraisal on how membrane trafficking may play an important role in the pathogenesis of the disease and how this could be exploited for effective therapy.     

Sorting of proteins to storage vacuoles: how many mechanisms?

Vacuoles receive their proteins through the secretory pathway, this requires protein sorting signals and molecular machineries that, until recently, have been believed to be markedly distinct for lytic and storage vacuoles. However, new biochemical, morphological and genetic data indicate that the only known class of vacuolar sorting receptors, believed to be specific for lytic vacuoles, might also be involved in the sorting of certain storage proteins. Furthermore, storage vacuoles can have a complex multimembrane structure that is difficult to explain based on a single trafficking mechanism. A new array of possible molecular interactions is thus emerging that no longer supports a clear-cut distinction between the two types of vacuoles based on sorting signals and putative receptors.     

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.     

Metal nanoclusters: Protein corona formation and implications for biological applications

Metal nanoclusters (NCs) are a new type of nanoprobe with great potential in various biological applications. For biocompatible and efficient utilization of NCs, a thorough understanding of their interactions with biological systems is highly important. Herein, we focus on recent studies addressing interactions between metal NCs and proteins as well as implications for their further biological application. These findings show that protein adsorption not only affects the photophysical properties of NCs, but also influences their subsequent biological behavior, i.e., cellular uptake and cytotoxicity. Moreover, specific protein–NC interactions have also been harnessed to develop novel protein discrimination strategies.     

On the lack of specificity of proteins and its consequences for a theory of biological organization

It is now widely recognized that gene expression and cellular processes include a probabilistic component. However, this does not essentially modify the theory of genetic programming. This stochastic aspect, which is called noise, is usually conceived as a margin of fluctuation in the way the genetic program functions and the latter remains understood as a specific mechanism guided by genetic information. In contrast, recent data show that proteins do not possess a high level of specificity. They can interact with numerous molecular partners. As a consequence molecular interactions are not simply “noisy”. Because they are subject to large combinatorial interaction possibilities, they are also intrinsically stochastic and must be sorted out by the cell structure. This contradicts the genetic programming theory which is based on the idea that protein interactions are directed by their stereospecificity and genetic information. Taking into account the lack of protein specificity leads to a new theory. Natural selection acts not only in evolution but also in ontogenesis by sorting stochastic molecular interactions. In this frame, the making up of an organism, instead of being a simple bottom-top process in which information flows from genes to phenotypes, is both a bottom-top and top-bottom process. Genes provide proteins, but their stochastic interactions are sorted by selective constraints arising from the cell and multi-cellular structures, which are themselves subject to the action of natural selection.     

Drosophila egg chamber elongation

As tissues and organs are formed, they acquire a specific shape that plays an integral role in their ability to function properly. A relatively simple system that has been used to examine how tissues and organs are shaped is the formation of an elongated Drosophila egg. While it has been known for some time that Drosophila egg elongation requires interactions between a polarized intracellular basal actin network and a polarized extracellular network of basal lamina proteins, how these interactions contribute to egg elongation remained unclear. Recent studies using live imaging have revealed two novel processes, global tissue rotation and oscillating basal actomyosin contractions, which have provided significant insight into how the two polarized protein networks cooperate to produce an elongated egg. This review summarizes the proteins involved in Drosophila egg elongation and how this recent work has contributed to our current understanding of how egg elongation is achieved.     

Drosophila egg chamber elongation: Insights into how tissues and organs are shaped

As tissues and organs are formed, they acquire a specific shape that plays an integral role in their ability to function properly. A relatively simple system that has been used to examine how tissues and organs are shaped is the formation of an elongated Drosophila egg. While it has been known for some time that Drosophila egg elongation requires interactions between a polarized intracellular basal actin network and a polarized extracellular network of basal lamina proteins, how these interactions contribute to egg elongation remained unclear. Recent studies using live imaging have revealed two novel processes, global tissue rotation and oscillating basal actomyosin contractions, which have provided significant insight into how the two polarized protein networks cooperate to produce an elongated egg. This review summarizes the proteins involved in Drosophila egg elongation and how this recent work has contributed to our current understanding of how egg elongation is achieved.     

BiP and zein binding domains within the delta zein protein

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.     

Amino acid contacts in proteins adapted to different temperatures: hydrophobic interactions and surface charges play a key role

Thermophiles, mesophiles, and psychrophiles have different amino acid frequencies in their proteins, probably because of the way the species adapt to very different temperatures in their environment. In this paper, we analyse how contacts between sidechains vary between homologous proteins from species that are adapted to different temperatures, but displaying relatively high sequence similarity. We investigate whether specific contacts between amino acids sidechains is a key factor in thermostabilisation in proteins. The dataset was divided into two subsets with optimal growth temperatures from 0–40 and 35–102°C. Comparison of homologues was made between low-temperature species and high-temperature species within each subset. We found that unspecific interactions like hydrophobic interactions in the core and solvent interactions and entropic effects at the surface, appear to be more important factors than specific contact types like salt bridges and aromatic clusters. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Apical transport of osteopontin is independent of N-glycosylation and sialylation

Studies of how epithelial surface polarity into apical and basolateral domains is generated and maintained have proposed that carbohydrate modifications serve as apical targeting signals for proteins by interacting with lectin sorters. However, the experimental evidence in support of N-glycans, O-glycans and sialic acids mediating apical transport is still very controversial. This could be partly due to the fact that in most studies exogenously expressed proteins were analysed. One has, therefore, examined the role of carbohydrate moieties in apical targeting of the endogenous secretory protein osteopontin in MDCK cells. It was found, however, that sorting of osteopontin does not require N-glycosylation of the protein itself nor that of other factors involved in the sorting process. Incubation of cells with the inhibitor of O-glycosylation benzyl- &#102 -GalNAc reduced the molecular weight of osteopontin by blocking sialic acid addition to O-glycans. Interestingly, also impairment of sialylation had no effect on polar secretion of the protein. Thus, the results show that both N-glycans and sialic acids are not essential sorting signals, suggesting that inner core carbohydrates and/or a proteinaceous signal mediate apical targeting of osteopontin.