Orientation of Small Multidrug Resistance Transporter Subunits in the Membrane: Correlation with the Positive-Inside Rule

Small multidrug resistance (SMR) transport proteins provide a model for the evolution of larger two-domain transport proteins. The orientation in the membrane of 27 proteins from the SMR family was determined using the reporter fusion technique. Nine members were encoded monocistronically (singles) and shown to insert in both orientations (dual topology). Eighteen members were encoded in pairs on the chromosome and shown to insert in fixed orientations; the two proteins in each pair invariably had opposite orientations in the membrane. Interaction between the two proteins in pairs was demonstrated by copurification. The orientation in the membrane of either protein in the pair was affected only marginally by the presence of the other protein.For the proteins in pairs, the orientation in the membrane correlated well with the distribution of positively charges residues (R + K) over the cytoplasmic and extracellular loops (positive-inside rule). In contrast, dual-topology insertion of the singles was predicted less well by the positive-inside rule. Three singles were predicted to insert in a single orientation with the N-terminus and the C-terminus at the extracellular side of the membrane. Analysis of charge distributions suggests the requirement of a threshold number of charges in the cytoplasmic loops for the positive-inside rule to be of predictive value. It is concluded that a combined analysis of gene organization on the chromosome and phylogeny is sufficient to distinguish between fixed or dual topology of SMR members and, probably, similar types of membrane proteins. The positive-inside rule can be used to predict the orientation of members in pairs, but is not suitable as a sole predictor of dual topology.     

Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

Cryo-EM insights into tail-anchored membrane protein biogenesis in eukaryotes

Tail-anchored (TA) proteins are a biologically significant class of membrane proteins, which require specialised cellular pathways to insert their single C-terminal transmembrane domain into the correct membrane. Cryo-electron microscopy has recently provided new insights into the organelle-specific machineries for TA protein biogenesis. Structures of targeting and insertase complexes within the canonical guided entry of TA proteins (GET) pathway indicate how substrates are faithfully chaperoned into the endoplasmic reticulum (ER) membrane in metazoans. The core of the GET insertase is conserved within structures of the ER membrane protein complex (EMC), which acts in parallel to insert a different subset of TA proteins. Furthermore, structures of the dislocases Spf1 and Msp1 show how they remove mislocalised TA proteins from the ER and outer mitochondrial membranes respectively.     

Transmission And Scanning Electron Microscope Preparations Of The Same Cell Culture

A technique has been developed which allows transmission electron microscopy and scanning electron microscopy to be performed on the same cell culture sample. The technique uses the Costar 3393 Leighton Tube containing a plastic insert, which does not stick to epoxy, for transmission electron microscopy. A cut piece of the plastic insert can be critical point dried, sputter coated and viewed under high vacuum with the scanning electron microscope.     

Structure of the armadillo repeat domain of plakophilin 1

The p120ctn subfamily of armadillo domain proteins has roles in modulating intercellular adhesion by cadherin-containing junctions. We have determined the crystal structure of the arm repeat domain from plakophilin-1 (PKP1), a member of the p120ctn subfamily that is found in desmosomes. The structure reveals that the domain has nine instead of the expected ten arm repeats. A sequence predicted to be an arm repeat is instead a large insert which serves as a wedge that produces a significant bend in the overall domain structure. Structure-based sequence alignments indicate that the nine repeats and large insert are common to this subfamily of armadillo proteins. A prominent basic patch on the surface of the protein may serve as a binding site for partners of these proteins.     

Transmission and scanning electron microscope preparations of the same cell culture.

A technique has been developed which allows transmission electron microscopy and scanning electron microscopy to be performed on the same cell culture sample. The technique uses the Costar 3,393 Leighton Tube containing a plastic insert, which does not stick to epoxy, for transmission electron microscopy. A cut piece of the plastic insert can be critical point dried, sputter coated and viewed under high vacuum with the scanning electron microscope.     

The saposin-like domain of the plant aspartic proteinase precursor is a potent inducer of vesicle leakage

A unique feature of plant aspartic proteinase precursors is the presence of an internal domain, known as plant-specific insert, whose function is not completely understood. The three-dimensional structure of the plant-specific insert resembles that of saposin-like proteins, a group of lipid-binding proteins involved in a variety of physiological processes. Here we show that recombinant plant-specific insert is able to interact with phospholipid vesicles and to induce leakage of their contents in a pH- and lipid-dependent manner. The leakage activity is higher at pH 4.5 and requires the presence of acidic phospholipids such as phosphatidylserine. To determine whether the same effect could be observed when the plant-specific insert is part of the precursor form, procardosin A and a mutant form lacking this specific domain were produced and characterized. Procardosin A displays a similar activity profile, whereas the mutant without the plant-specific insert shows only residual activity. These findings indicate that the plant-specific insert domain of plant aspartic proteinases mediates an interaction of their precursors with phospholipid membranes and induces membrane permeabilization. It is therefore possible that the plant-specific insert, alone or in conjunction with the proteolytic activity of plant aspartic proteinases, may function either as a defensive weapon against pathogens or in late autolysis of plant cells.     

Vibrational fluctuations of hydrogen bonds in a DNA double helix with alternating TA type inserts.

The Green's function technique is applied to a study of breathing modes in a DNA double helix which contains a region of different base pairs from the rest of the double helix. The calculation is performed on an alternating poly(dC-dG).poly(dC-dG) helix in the B conformation with four consecutive base pairs replaced by a model of a biological promoter region with four alternating T-A,A-T base pairs, henceforth referred to as (TATA)2. The average stretch of interbase hydrogen bonds is found to be amplified around the insert. This is likely related to the (TATA)2 insert having a lower stability against hydrogen bond melting than the two semi-infinite poly(dC-dG).poly(dC-dG) helices. The insert region may be considered to be a site of enhanced tendency to melt in such a helix. The results show that an alternating AT insert of four base pairs has a larger average hydrogen bond stretch inside and outside the insert region than the average hydrogen bond stretch inside and outside an insert of four consecutive A-T base pairs, henceforth referred to as (AAAA).(TTTT). Calculations are performed which show that the enhancement of the average hydrogen bond stretch around an alternating TA type insert is greatly dependent upon the local modes and not the inband modes. The amount of local mode enhanced average stretch is explored as a function of insert size.     

The 2.2 A resolution crystal structure of Bacillus cereus Nif3-family protein YqfO reveals a conserved dimetal-binding motif and a regulatory domain

YqfO of Bacillus cereus is a member of the widespread Nif3 family of proteins, which has been highlighted as an important target for structural genomics. The N- and C-terminal domains are conserved across the family and contain a dimetal-binding motif in a putative active site. YqfO contains an insert in the middle of the protein, present in a minority of bacterial family members. The structure of YqfO was determined at a resolution of 2.2 A and reveals conservation of the putative active site. It also reveals the previously unknown structure of the insert, which despite extremely limited sequence conservation, bears great similarity to PII, CutA, and a number of other trimeric regulatory proteins. Our results suggest that this domain acts as a signal sensor to regulate the still-unknown catalytic activity of the more-conserved domains.     

The Insert Region of RhoA Is Essential for Rho Kinase Activation and Cellular Transformation

RhoA is involved in multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These processes are mediated by a variety of downstream effector proteins. However, which effectors are involved in cellular transformation and how these proteins are activated following interaction with Rho remains to be established. A unique feature that distinguishes the Rho family from other Ras-related GTPases is the insert region, which may confer Rho-specific signaling events. Here we report that deletion of the insert region does not result in impaired effector binding. Instead, this insert deletion mutant (RhoDeltaRas, in which the insert helix has been replaced with loop 8 of Ras) acted in a dominant inhibitory fashion to block RhoA-induced transformation. Since RhoDeltaRas failed to promote stress fiber formation, we examined the ability of this mutant to bind to and subsequently activate Rho kinase. Surprisingly, RhoDeltaRas-GTP coprecipitated with Rho kinase but failed to activate it in vivo. These data suggested that the insert domain is not required for Rho kinase binding but plays a role in its activation. The constitutively active catalytic domain of Rho kinase did not promote focus formation alone or in the presence of Raf(340D) but cooperated with RhoDeltaRas to induce cellular transformation. This suggests that Rho kinase needs to cooperate with additional Rho effectors to promote transformation. Further, the Rho kinase catalytic domain reversed the inhibitory effect of RhoDeltaRas on Rho-induced transformation, suggesting that one of the downstream targets of Rho-induced transformation abrogated by RhoDeltaRas is indeed Rho kinase. In conclusion, we have demonstrated that the insert region of RhoA is required for Rho kinase activation but not for binding and that this kinase activity is required to induce morphologic transformation of NIH 3T3 cells.     

A general insert label for peptide display on chimeric filamentous bacteriophages

The foreign insert intended to be displayed via recombinant phage proteins can have a negative effect on protein expression and phage assembly. A typical example is the case of display of peptides longer than 6 amino acid residues on the major coat protein, protein VIII of the filamentous bacteriophages M13 and fd. A solution to this problem has been the use of "two-gene systems" generating chimeric phages that concomitantly express wild-type protein VIII along with recombinant protein VIII. Although the two-gene systems are much more permissive in regard to insert length and composition, some cases can still adversely affect phage assembly. Although these phages genotypically contain the desired DNA of the insert, they appear to be phenotypically wild type. To avoid false-negative results when using chimeric phages in binding studies, it is necessary to confirm that the observed lack of phage recognition is not due to faulty assembly and display of the intended insert. Here we describe a strategy for generating antibodies that specifically recognize recombinant protein VIII regardless of the nature of its foreign insert. These antibodies can be used as a general monitor of the display of recombinant protein VIII into phage particles.     

Oligonucleotide-mediated, PCR-independent cloning by homologous recombination

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).     

Structure and mechanism of the saposin-like domain of a plant aspartic protease

Many plant aspartic proteases contain an additional sequence of ∼100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plant-specific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å, revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus.     

PRALINETM: a strategy for improved multiple alignment of transmembrane proteins

MOTIVATION: Membrane-bound proteins are a special class of proteins. The regions that insert into the cell-membrane have a profoundly different hydrophobicity pattern compared with soluble proteins. Multiple alignment techniques use scoring schemes tailored for sequences of soluble proteins and are therefore in principle not optimal to align membrane-bound proteins. RESULTS: Transmembrane (TM) regions in protein sequences can be reliably recognized using state-of-the-art sequence prediction techniques. Furthermore, membrane-specific scoring matrices are available. We have developed a new alignment method, called PRALINETM, which integrates these two features to enhance multiple sequence alignment. We tested our algorithm on the TM alignment benchmark set by Bahr et al. (2001), and showed that the quality of TM alignments can be significantly improved compared with the quality produced by a standard multiple alignment technique. The results clearly indicate that the incorporation of these new elements into current state-of-the-art alignment methods is crucial for optimizing the alignment of TM proteins. AVAILABILITY: A webserver is available at http://www.ibi.vu.nl/programs/pralinewww.     

Molecular signatures for the main phyla of photosynthetic bacteria and their subgroups

The bacterial groups corresponding to different photosynthetic prokaryotes are presently identified mainly on the basis of their branching in phylogenetic trees. The availability of genome sequences is enabling identification of many molecular signatures that are specific for different groups of photosynthetic bacteria. Our recent work has identified large numbers of signatures consisting of conserved inserts or deletions (indels) in widely distributed proteins, as well as whole proteins that are specific for various sequenced species/strains from Cyanobacteria, Chlorobi, and Proteobacteria phyla. Based upon these signatures, it is now possible to identify/distinguish bacteria from these phyla of photosynthetic bacteria as well as their major subclades in clear molecular terms. The use of these signatures in conjunction with phylogenomic analyses, summarized here, is leading to a holistic picture concerning the branching order and evolutionary relationships among the above groups of photosynthetic bacteria. Although detailed studies in this regard have not yet been carried on Chloroflexi and Heliobacteriaceae, we have identified some conserved indels that are specific for these groups. Some of the conserved indels for the photosynthetic bacteria are present in photosynthesis-related proteins. These include a 4 aa insert in the pyruvate flavodoxin/ferridoxin oxidoreductase that is specific for the genus Chloroflexus, a 2 aa insert in magnesium chelatase that is uniquely shared by all Cyanobacteria except the deepest branching Clade A (Gloebacterales), a 6 aa insert in an A-type flavoprotein that is specific for various marine unicellular Cyanobacteria, a 2 aa insert in heme oxygenase that is specific for various Prochlorococcus strains/isolates, and 1 aa deletion in the protein protochlorophyllide oxidoreductase that is commonly shared by various Prochlorococcus strains except the deepest branching isolates MIT 9303 and MIT 9313. The identified CSIs are located in the structures of these proteins in surface loops indicating that they may be important in mediating protein–protein interactions. The cellular functions of these conserved indels, or most of the signature proteins are presently unknown, but they provide valuable means for discovering novel properties that are unique to different groups of photosynthetic bacteria.     

In Silico Insights into Protein-Protein Interactions and Folding Dynamics of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.     

α-Synemin localizes to the M-band of the sarcomere through interaction with the M10 region of titin

α-Synemin contains a unique 312 amino acid insert near the end of its C-terminal tail. Therefore we set out to determine if the insert is a site of protein–protein interaction that regulates the sub-cellular localization of this large isoform of synemin. Yeast-two hybrid analysis indicated that this region is a binding site for the M10 region of titin. This was confirmed with GST pull-down assays. Co-immunoprecipitation of endogenous proteins indicated close association of the two proteins in vivo and immunostaining of cardiomyocytes demonstrated co-localization of the proteins at the M-band of the sarcomere.