Topogenic signals in integral membrane proteins

Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic.proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information.     

Anionic phospholipids are determinants of membrane protein topology.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.     

Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p

Mgm1p is a nuclearly encoded GTPase important for mitochondrial fusion. Long and short isoforms of the protein are generated in a unique "alternative topogenesis" process in which the most N-terminal of two hydrophobic segments in the protein is inserted into the inner mitochondrial membrane in about half of the molecules and translocated across the inner membrane in the other half. In the latter population, the second hydrophobic segment is cleaved by the inner membrane protease Pcp1p, generating the short isoform. Here, we show that charged residues in the regions flanking the first segment critically affect the ratio between the two isoforms, providing new insight into the importance of charged residues in the insertion of proteins into the mitochondrial inner membrane.     

Yeast Oxa1 interacts with mitochondrial ribosomes: the importance of the C-terminal region of Oxa1

The yeast mitochondrial Oxa1 protein is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1 mediates the insertion of proteins (nuclearly and mitochondrially encoded) into the inner membrane. The mitochondrially encoded substrates interact directly with Oxa1 during their synthesis as nascent chains and in a manner that is supported by the associated ribosome. We have investigated if the Oxa1 complex interacts with the mitochondrial ribosome. Evidence to support a physical association between Oxa1 and the large ribosomal subunit is presented. Our data indicate that the matrix-exposed C-terminal region of Oxa1 plays an important role supporting the ribosomal-Oxa1 interaction. Truncation of this C-terminal segment compromises the ability of Oxa1 to support insertion of substrate proteins into the inner membrane. Oxa1 can be cross-linked to Mrp20, a component of the large ribosomal subunit. Mrp20 is homologous to L23, a subunit located next to the peptide exit tunnel of the ribosome. We propose that the interaction of Oxa1 with the ribosome serves to enhance a coupling of translation and membrane insertion events.     

A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain

The orientation of membrane proteins is determined by the asymmetric distribution of charged residues in the sequences flanking the transmembrane domains. For the inner membrane of Escherichia coli, numerous studies have shown that an excess of positively charged residues defines a cytoplasmic domain of a membrane protein ("positive inside" rule). The role of negatively charged residues in establishing membrane protein topology, however, is not completely understood. To investigate the influence of negatively charged residues on this process in detail, we have constructed a single spanning chimeric receptor fragment comprising the N terminus and first transmembrane domain of the heptahelical G protein-coupled vasopressin V(2) receptor and the first cytoplasmic loop of the beta(2)-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane of E. coli with its N terminus facing the cytoplasm (N(in)-C(out) orientation), although both membrane-flanking domains had rather similar topogenic determinants. The orientation of the receptor fragment was changed after the introduction of single glutamate residues into the N terminus. Orientation inversion, however, was found to be dependent on the location of the glutamate substitutions, which had to lie within a narrow window up to 6 residues distant from the transmembrane domain. These results demonstrate that a single negatively charged residue can play an active role as a topogenic determinant of membrane proteins in the inner membrane of E. coli, but only if it is located adjacent to a transmembrane domain.     

Mitochondrial processing peptidases

Three peptidases are responsible for the proteolytic processing of both nuclearly and mitochondrially encoded precursor polypeptides targeted to the various subcompartments of the mitochondria. Mitochondrial processing peptidase (MPP) cleaves the vast majority of mitochondrial proteins, while inner membrane peptidase (IMP) and mitochondrial intermediate peptidase (MIP) process specific subsets of precursor polypeptides. All three enzymes are structurally and functionally conserved across species, and their human homologues begin to be recognized as potential players in mitochondrial disease.     

Voltage-dependent orientation of membrane proteins

In order to study the influence of electrostatic forces on the disposition of proteins in membranes, we have examined the interaction of a receptor protein and of a membrane-active peptide with black lipid membranes. In the first study we show that the hepatic asialoglycoprotein receptor can insert spontaneously into lipid bilayers from the aqueous medium. Under the influence of a trans-positive membrane potential, the receptor, a negatively charged protein, appears to change its disposition with respect to the membrane. In the second study we consider melittin, an amphipathic peptide containing a generally hydrophobic stretch of 19 amino acids followed by a cluster of four positively charged residues at the carboxy terminus. The hydrophobic region contains two positively charged residues. In response to trans-negative electrical potential, melittin appears to assume a transbilayer position. These findings indicate that electrostatic forces can influence the disposition, and perhaps the orientation, of membrane proteins. Given the inside-negative potential of most or all cells, we would expect transmembrane proteins to have clusters of positively charged residues adjacent to the cytoplasmic ends of their hydrophobic transmembrane segments, and clusters of negatively charged residues just to the extracytoplasmic side. This expectation has been borne out by examination of the few transmembrane proteins for which there is sufficient information on both sequence and orientation. Surface and dipole potentials may similarly affect the orientation of membrane proteins.     

Intrinsically disordered regions have specific functions in mitochondrial and nuclear proteins

Proteins in general consist not only of globular structural domains (SDs), but also of intrinsically disordered regions (IDRs), i.e. those that do not assume unique three-dimensional structures by themselves. Although IDRs are especially prevalent in eukaryotic proteins, the functions are mostly unknown. To elucidate the functions of IDRs, we first divided eukaryotic proteins into subcellular localizations, identified IDRs by the DICHOT system that accurately divides entire proteins into SDs and IDRs, and examined charge and hydropathy characteristics. On average, mitochondrial proteins have IDRs more positively charged than SDs. Comparison of mitochondrial proteins with orthologous prokaryotic proteins showed that mitochondrial proteins tend to have segments attached at both N and C termini, high fractions of which are IDRs. Segments added to the N-terminus of mitochondrial proteins contain not only signal sequences but also mature proteins and exhibit a positive charge gradient, with the magnitude increasing toward the N-terminus. This finding is consistent with the notion that positively charged residues are added to the N-terminus of proteobacterial proteins so that the extended proteins can be chromosomally encoded and efficiently transported to mitochondria after translation. By contrast, nuclear proteins generally have positively charged SDs and negatively charged IDRs. Among nuclear proteins, DNA-binding proteins have enhanced charge tendencies. We propose that SDs in nuclear proteins tend to be positively charged because of the need to bind to negatively charged nucleotides, while IDRs tend to be negatively charged to interact with other proteins or other regions of the same proteins to avoid premature proteasomal degradation.     

Characterization of peptides released from mitochondria: evidence for constant proteolysis and peptide efflux

Conserved ATP-dependent proteases ensure the quality control of mitochondrial proteins and control essential steps in mitochondrial biogenesis. Recent studies demonstrated that non-assembled mitochondrially encoded proteins are degraded to peptides and amino acids that are released from mitochondria. Here, we have characterized peptides extruded from mitochondria by mass spectrometry and identified 270 peptides that are exported in an ATP- and temperature-dependent manner. The peptides originate from 51 mitochondrially and nuclearly encoded proteins localized mainly in the matrix and inner membrane, indicating that peptides generated by the activity of all known mitochondrial ATP-dependent proteases can be released from the organelle. Pulse-labeling experiments in logarithmically growing yeast cells revealed that approximately 6-12% of preexisting and newly imported proteins is degraded and contribute to this peptide pool. Under respiring conditions, we observed an increased proteolysis of newly imported proteins that suggests a higher turnover rate of respiratory chain components and thereby rationalizes the predominant appearance of representatives of this functional class in the detected peptide pool. These results demonstrated a constant efflux of peptides from mitochondria and provided new insight into the stability of the mitochondrial proteome and the efficiency of mitochondrial biogenesis.     

Membrane protein insertion of variant MscL proteins occurs at YidC and SecYEG of Escherichia coli

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypoosmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon but requires YidC. Depletion of YidC inhibits translocation of the protein across the membrane. Insertion of MscL occurs primarily in a proton motive force-independent manner. The hydrophilic loop region of MscL has 29 residues that include 5 charged residues. Altering the charges in the periplasmic loop of MscL affects the requirements for membrane insertion. The introduction of one, two or three negatively charged amino acids makes the insertion dependent on the electrochemical membrane potential and gradually dependent on the Sec translocon, whereas the addition of five negatively charged residues as well as the addition of three positively charged residues inhibits membrane insertion of MscL. However, we find that the mutant with three uncharged residues requires both the SecYEG complex and YidC but not SecA for membrane insertion. In vivo cross-linking data showed that the newly synthesized MscL interacts with YidC and with SecY. Therefore, the MscL mutants use a membrane insertion mechanism that involves SecYEG and YidC simultaneously.     

Topology of eukaryotic type II membrane proteins: importance of N-terminal positively charged residues flanking the hydrophobic domain

We have tested the role of different charged residues flanking the sides of the signal/anchor (S/A) domain of a eukaryotic type II (N(cyt)C(exo)) integral membrane protein in determining its topology. The removal of positively charged residues on the N-terminal side of the S/A yields proteins with an inverted topology, while the addition of positively charged residues to only the C-terminal side has very little effect on orientation. Expression of chimeric proteins composed of domains from a type II protein (HN) and the oppositely oriented membrane protein M2 indicates that the HN N-terminal domain is sufficient to confer a type II topology and that the M2 N-terminal ectodomain can direct a type II topology when modified by adding positively charged residues. These data suggest that eukaryotic membrane protein topology is governed by the presence or absence of an N-terminal signal for retention in the cytoplasm that is composed in part of positive charges.     

Membrane protein topology: effects of delta mu H+ on the translocation of charged residues explain the ''positive inside'' rule.

The membrane electrochemical potential is critical for the export of most periplasmic proteins in Escherichia coli. Its exact role during insertion of integral inner membrane proteins, however, remains obscure. Using derivatives of the inner membrane protein leader peptidase (Lep), we now show that the membrane potential appears to stimulate the membrane translocation of chain segments containing negatively charged residues, that positively charged regions appear to be more easily translocated in the absence of a potential, and that certain Lep constructs insert with different topologies in the presence and absence of a membrane potential, suggesting that the electrochemical potential introduces an asymmetry between the topological effects of positively and negatively charged amino acids during the process of membrane protein insertion in E. coli.     

Proline residues of transmembrane domains determine the sorting of inner membrane proteins in mitochondria

Most inner membrane proteins of mitochondria are synthesized in the cytosol and reach the inner membrane using one of two alternative sorting pathways. On the stop transfer route, proteins are arrested during import at the level of the inner membrane. The conservative sorting pathway involves translocation through the inner membrane and insertion from the matrix. It is unclear how the translocase of the inner membrane 23 protein translocation machinery differentiates between the two classes of proteins. Here we show that proline residues in hydrophobic stretches strongly disfavor the translocation arrest of transmembrane domains (TMDs) and favor the transfer of preproteins to the matrix. We propose that proline residues, together with the hydrophobicity of the TMD and the presence of charged residues COOH-terminally flanking the TMD, are determinants of the intramitochondrial sorting of inner membrane proteins.     

Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA

Oxa1p is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1p has been shown previously to directly facilitate the export of the N-terminal domains of membrane proteins across the inner membrane to the intermembrane space of mitochondria. Here we report on a general role of Oxa1p in the membrane insertion of proteins. (i) The function of Oxa1p is not limited to the insertion of membrane proteins that undergo N-terminal tail export; rather, it also extends to the insertion of other polytopic proteins such as the mitochondrially encoded Cox1p and Cox3p proteins. These are proteins whose N-termini are retained in the mitochondrial matrix. (ii) Oxa1p interacts directly with these substrates prior to completion of their synthesis. (iii) The interaction of Oxa1p with its substrates is particularly strong when nascent polypeptide chains are inserted into the inner membrane, suggesting a direct function of Oxa1p in co-translational insertion from the matrix. Taken together, we conclude that the Oxa1 complex represents a general membrane protein insertion machinery in the inner membrane of mitochondria.     

Mitochondrially encoded methionine is inversely related to longevity in mammals

Methionine residues in proteins react readily with reactive oxygen species making them particularly sensitive to oxidation. However, because oxidized methionine can be reduced back in a catalyzed reaction, it has been suggested that methionine residues act as oxidant scavengers, protecting not only the proteins where they are located but also the surrounding macromolecules. To investigate whether methionine residues may be selected for or against animal longevity, we carried out a meta-examination of mitochondrial genomes from mammalian species. Our analyses unveiled a hitherto unnoticed observation: mitochondrially encoded polypeptides from short-lived species are enriched in methionine when compared with their long-lived counterparts. We show evidence suggesting that methionine addition to proteins in short-lived species, rather than methionine loss from proteins in long-lived species, is behind the reported difference in methionine usage. The inverse association between longevity and methionine, which persisted after correction for body mass and phylogenetic interdependence, was paralleled by the methionine codon AUA, but not by the codon AUG. Although nuclear encoded mitochondrial polypeptides exhibited higher methionine usage than nonmitochondrial proteins, correlation with longevity was only found within the group of those polypeptides located in the inner mitochondrial membrane. Based on these results, we propose that short-lived animals subjected to higher oxidative stress selectively accumulate methionine in their mitochondrially encoded proteins, which supports the role of oxidative damage in aging.     

Different positively charged amino acids have similar effects on the topology of a polytopic transmembrane protein in Escherichia coli.

Integral membrane proteins from a wide variety of sources conform to a "positive-inside rule," with many more positively charged amino acids in their cytoplasmic as compared to extracytoplasmic domains. A growing body of experimental work also points to positively charged residues in regions flanking the apolar transmembrane segments as being the main topological determinants. In this paper, we report a systematic comparison of the effects of positively (Arg, Lys, His) as well as negatively (Asp, Glu) charged residues on the membrane topology of a model Escherichia coli inner membrane protein. Our results show that positive charge is indeed the major factor determining the transmembrane topology, with Arg and Lys being of nearly equal efficiency. His, although normally a very weak topological determinant, can be potentiated by a lowering of the cytoplasmic pH. Asp and Glu affect the topology to similar extents and only when present in very high numbers.     

An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.     

Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors.

The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties. We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site. Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region. These residues appear to inhibit proper insertion of pIII into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles. Suppressor mutations in the prlA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues. We conclude that insertion of pIII fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism. The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries.     

Initial steps in protein membrane insertion. Bacteriophage M13 procoat protein binds to the membrane surface by electrostatic interaction.

Bacteriophage M13 procoat protein is synthesized on free polysomes prior to its assembly into the inner membrane of Escherichia coli. As an initial step of the membrane insertion pathway, the precursor protein interacts with the cytoplasmic face of the inner membrane. We have used oligonucleotide-directed mutagenesis to study the regions of the procoat protein involved in membrane binding. We find that there is an absolute requirement for positively charged amino acids at both ends of the protein. Replacing these with negatively charged residues resulted in an accumulation of the precursor in the cytoplasm. We propose that the positively charged amino acids are directly involved in membrane binding, possibly directly to the negatively charged phospholipid head groups. This was tested in vitro with artificial liposomes. Whereas wild-type procoat interacted with these liposomes, we found that procoat mutants with negatively charged amino acids at both ends did not bind. Therefore, we conclude that newly synthesized M13 procoat protein binds electrostatically to the negatively charged inner membrane of E. coli.     

Mode of interaction between lysozyme and the other proteins of the hen's egg vitelline membrane

Salt solutions and charged detergents are efficient solubilizing agents for ovovitelline membrane lysozyme. Reassociation experiments with chemically modified lysozymes indicate that positively charged amino acid residues of lysozyme (the epsilon-amino group of lysine and the guanidino group of arginine) are involved in the interaction with other proteins of the vitelline membrane. Exogenous proteins are adsorbed to lysozyme-free vitelline membranes, only if they have a high pI, comparable to that of lysozyme. It is concluded that the lysozyme-ovovitelline membrane interaction is predominantly ionic. An ovomucin-lysozyme complex is postulated as the major component of the outer layer of the membrane.