Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

The Fascinating Coat Surrounding Mycobacteria

The mycobacterial cell envelope is fascinating in several ways. First, its composition is unique by the exceptional lipid content, which consists of very long-chain (up to C90) fatty acids, the so-called mycolic acids, and a variety of exotic compounds. Second, these lipids are atypically organized into a Gram-negative-like outer membrane (mycomembrane) in these Gram-positive bacteria, as recently revealed by CEMOVIS, and this mycomembrane also contains pore-forming proteins. Third, the mycolic acids esterified a holistic heteropolysaccharide (arabinogalacan), which in turn is linked to the peptidoglycan to form the cell wall skeleton (CWS). In slow-growing pathogenic mycobacterial species, this giant structure is surrounded by a capsular layer composed mainly of polysaccharides, primarily a glycogen-like glucan. The CWS is separated from the plasma membrane by a periplasmic space. A challenging research avenue for the next decade comprises the identification of the components of the uptake and secretion machineries and the isolation and biochemical characterization of the mycomembrane.

Characterization of the Agrobacterium tumefaciens VirB2 pilin of the VirB/D4 Type IV Secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) delivers oncogenic T-DNA and effector proteins to susceptible plant cells. This leads to the formation of tumors termed Crown Galls. The VirB/D4 T4SS is comprised of 12 subunits (VirB1 to VirB11 and VirD4), which assemble to form two structures, a secretion channel spanning the cell envelope and a T-pilus extending from the cell surface. In A. tumefaciens, the VirB2 pilin subunit is required for assembly of the secretion channel and is the main subunit of the T-pilus. The focus of this thesis is to define key reactions associated with the T4SS biogenesis pathway involving the VirB2 pilin. Topology studies demonstrated that VirB2 integrates into the inner membrane with two transmembrane regions, a small cytoplasmic loop, and a long periplasmic loop comprised of covalently linked N and C termini. VirB2 was shown by the substituted cysteine accessibility method (SCAM) to adopt distinct structural states when integrated into the inner membrane and when assembled as a component of the secretion channel and the T-pilus. The VirB4 and VirB11 ATPases were shown by SCAM to modulate the structural state of membrane-integrated VirB2 pilin, and evidence was also obtained that VirB4 mediates extraction of pilin from the membrane. A model that VirB4 functions as a pilin dislocase by an energy-dependent mechanism was further supported by coimmunoprecipitation and osmotic shock studies. Mutational studies identified two regions of VirB10, an N-terminal transmembrane domain and an outer membrane-associated domain termed the antennae projection, that contribute selectively to T-pilus biogenesis. Lastly, characterization of a VirB10 mutant that confers a ‘leaky’ channel phenotype further highlighted the role of VirB10 in gating substrate translocation across the outer membrane as well as T-pilus biogenesis. Results of my studies support a working model in which the VirB4 ATPase catalyzes dislocation of membrane-integrated pilin, and distinct domains of VirB10 coordinate pilin incorporation into the secretion channel and the extracellular T-pilus.

Different dark conformations function in color-sensitive photosignaling by the sensory rhodopsin I-HtrI complex.

The haloarchaeal phototaxis receptor sensory rhodopsin I (SRI) in complex with its transducer HtrI delivers an attractant signal from excitation with an orange photon and a repellent signal from a second near-UV photon excitation. Using a proteoliposome system with purified SRI in complex with its transducer HtrI, we identified by site-directed fluorescence labeling a site (Ser(155)) on SRI that is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser(155)Cys with a nitroxide side chain, we detected a change in conformation following one-photon excitation such that the spin probe exhibits a splitting of the outer hyperfine extrema (2A'(zz)) significantly smaller than that of the electron paramagnetic resonance spectrum in the dark state. The dark conformations of five mutant complexes that do not discriminate between orange and near-UV excitation show shifts to lower or higher 2A'(zz) values correlated with the alterations in their motility behavior to one- and two-photon stimuli. These data are interpreted in terms of a model in which the dark complex is populated by two conformers in the wild type, one that inhibits the CheA kinase (A) and the other that activates it (R), shifted in the dark by mutations and shifted in the wild-type SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.

A Schiff base connectivity switch in sensory rhodopsin signaling.

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI-HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.

Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive inside rule.

Transmembrane domain orientation within some membrane proteins is dependent on membrane lipid composition. Initial orientation occurs within the translocon, but final orientation is determined after membrane insertion by interactions within the protein and between lipid headgroups and protein extramembrane domains. Positively and negatively charged amino acids in extramembrane domains represent cytoplasmic retention and membrane translocation forces, respectively, which are determinants of protein orientation. Lipids with no net charge dampen the translocation potential of negative residues working in opposition to cytoplasmic retention of positive residues, thus allowing the functional presence of negative residues in cytoplasmic domains without affecting protein topology.

Intact flagellar motor of Borrelia burgdorferi revealed by cryo-electron tomography: evidence for stator ring curvature and rotor/C-ring assembly flexion.

The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of approximately 1,280 flagellar motors, a approximately 3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.

Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Functional proteomics and biochemsitry: working together to unravel mitochondrial phenomena of African trypansomes

Eukaryotic cells are compartmentalized into membrane-bound organelles in order to provide sheltered reaction rooms for various specific processes. Organelles are not randomly distributed in a cell or operate isolated from each other. At the contrary — some organelles are closely linked and their functions are tightly orchestrated. The most well-known example of two such organelles acting in concert are the ER and the mitochondrion that work together in order to coordinate cellular lipid biosynthesis, maintain Ca2+-homeostasis, regulate mitochondrial division and control mitochondrial/ER shape as well as to synchronize the movement of these organelles within a cell. To study the mitochondrion and its interface to the ER requires a simplified mitochondrial system. African trypanosomes represent such a system. The unicellular parasite that causes devastating diseases in humans and animals has only one large mitochondrion that does not undergo fission/fusion events except for the context of cell division. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the protozoan. Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal MOM proteome consists of 82 proteins, two thirds of which have never been associated with mitochondria before. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. Interestingly, a putative tethering complex in mammalian cells was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating, if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved. This ER-subdomain associated with mitochondria has been termed mitochondria-associated ER-membranes or MAM. The MAM has recently been implicated to play a key role in Alzheimer’s disease. It is therefore of broad and general interest to establish other eukaryotic model systems in order to investigate the MAM-MOM connection in more detail.

Functional proteomics and biochemsitry: working together to unravel mitochondrial phenomena of African trypansomes

Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of the mitochondrial model organism T. brucei and characterized its proteome. Our results show that the trypanosomal MOM proteome consists of 82 proteins. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. In mammalian cells, a putative tethering complex was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved.

Rapid method to express and purify human membrane protein using the Xenopus oocyte system for functional and low-resolution structural analysis.

Progress toward elucidating the 3D structures of eukaryotic membrane proteins has been hampered by the lack of appropriate expression systems. Recent work using the Xenopus oocyte as a novel expression system for structural analysis demonstrates the capability of providing not only the significant amount of protein yields required for structural work but also the expression of eukaryotic membrane proteins in a more native and functional conformation. There is a long history using the oocyte expression system as an efficient tool for membrane transporter and channel expression in direct functional analysis, but improvements in robotic injection systems and protein yield optimization allow the rapid scalability of expressed proteins to be purified and characterized in physiologically relevant structural states. Traditional overexpression systems (yeast, bacteria, and insect cells) by comparison require chaotropic conditions over several steps for extraction, solubilization, and purification. By contrast, overexpressing within the oocyte system for subsequent negative-staining transmission electron microscopy studies provides a single system that can functionally assess and purify eukaryotic membrane proteins in fewer steps maintaining the physiological properties of the membrane protein.

Functional proteomics and biochemistry: working together to unravel mitochondrial phenomena of African trypanosomes

Eukaryotic cells are compartmentalized into membrane-bound organelles in order to provide sheltered reaction rooms for various specific processes. Organelles are not randomly distributed in a cell or operate isolated from each other. At the contrary — some organelles are closely linked and their functions are tightly orchestrated. The most well-known example of two such organelles acting in concert are the ER and the mitochondrion that work together in order to coordinate cellular lipid biosynthesis, maintain Ca2+-homeostasis, regulate mitochondrial division and control mitochondrial/ER shape as well as to synchronize the movement of these organelles within a cell. To study the mitochondrion and its interface to the ER requires a simplified mitochondrial system. African trypanosomes represent such a system. The unicellular parasite that causes devastating diseases in humans and animals has only one large mitochondrion that does not undergo fission/fusion events except for the context of cell division. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the protozoan. Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal MOM proteome consists of 82 proteins, two thirds of which have never been associated with mitochondria before. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. Interestingly, a putative tethering complex in mammalian cells was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating, if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved. This ER-subdomain associated with mitochondria has been termed mitochondria-associated ER-membranes or MAM. The MAM has recently been implicated to play a key role in Alzheimer’s disease. It is therefore of broad and general interest to establish other eukaryotic model systems in order to investigate the MAM-MOM connection in more detail.