Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane.

Synaptobrevin/vesicle-associated membrane protein is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is proposed to provide specificity for the targeting and fusion of vesicles with the plasma membrane. It belongs to a class of membrane proteins which lack a signal sequence and contain a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored). We show that in neuroendocrine PC12 cells, synaptobrevin is not directly incorporated into the target organelle, synaptic-like vesicles. Rather, it is first inserted into the endoplasmic reticulum (ER) membrane and is then transported via the Golgi apparatus. Its insertion into the ER membrane in vitro occurs post-translationally, is dependent on ATP and results in a trans-membrane orientation of the hydrophobic tail. Membrane integration requires ER protein(s) different from the translocation components needed for proteins with signal sequences, thus suggesting a novel mechanism of insertion.     

Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.     

The endoplasmic reticulum (ER) translocon can differentiate between hydrophobic sequences allowing signals for glycosylphosphatidylinositol anchor addition to be fully translocated into the ER lumen

The signal sequence within polypeptide chains that designates whether a protein is to be anchored to the membrane by a glycosylphosphatidylinositol (GPI) anchor is characterized by a carboxyl-terminal hydrophobic domain preceded by a short hydrophilic spacer linked to the GPI anchor attachment (omega) site. The hydrophobic domain within the GPI anchor signal sequence is very similar to a transmembrane domain within a stop transfer sequence. To investigate whether the GPI anchor signal sequence is translocated across or integrated into the endoplasmic reticulum membrane we studied the translocation, GPI anchor addition, and glycosylation of different variants of a model GPI-anchored protein. Our results unequivocally demonstrated that the hydrophobic domain within a GPI signal cannot act as a transmembrane domain and is fully translocated even when followed by an authentic charged cytosolic tail sequence. However, a single amino acid change within the hydrophobic domain of the GPI-signal converts it into a transmembrane domain that is fully integrated into the endoplasmic reticulum membrane. These results demonstrated that the translocation machinery can recognize and differentiate subtle changes in hydrophobic sequence allowing either full translocation or membrane integration.     

Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon.

The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH(2)-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH(2)-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.     

Recognition of the carboxyl-terminal signal for GPI modification requires translocation of its hydrophobic domain across the ER membrane

A carboxyl-terminal hydrophobic domain is an essential component of the processed signal for attachment of the glycosyl-phosphatidylinositol (GPI) membrane anchor to proteins and it is linked to the site (omega) of GPI modification by a spacer domain. This study was designed to test the hypothesis that the hydrophobic domain interacts with the lipid bilayer of the endoplasmic reticulum (ER) membrane to optimally position the omega site for GPI modification. The hydrophobic domain of the GPI signal in the human folate receptor (FR) type alpha was substituted with the carboxyl-terminal segment of the low-density lipoprotein receptor (LDLR), including its membrane spanning region, without altering either the spacer or the omega site. The FR-alpha/LDLR chimera was not GPI modified but was attached to the plasma membrane by a polypeptide anchor. When the carboxyl-terminal half of the hydrophobic transmembrane polypeptide in the FR-alpha/LDLR chimera was altered by introduction of negatively charged (Asp) residues, or when the cytosolic domain in the chimera was deleted, the mutated proteins became GPI-anchored. On the other hand, attachment of a carboxyl-terminal segment of LDLR including the entire cytosolic domain to FR-alpha converted it into a transmembrane protein. The results indicate that in the FR-alpha/LDLR chimera the inability of the cellular machinery for GPI modification to recognize the hydrophobic domain is not due to the intrinsic nature of the peptide, but is rather due to the retention of the peptide within the lipid bilayer. It follows that the hydrophobic domain in the signal for GPI modification must traverse the ER membrane prior to recognition of the omega site by the GPI-protein transamidase. The results thus establish a critical topographical requirement for recognition of the GPI signal in the ER.     

An Artificial Mitochondrial Tail Signal/Anchor Sequence Confirms a Requirement for Moderate Hydrophobicity for Targeting

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.     

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.     

Deletion of the amino-terminal domain of asialoglycoprotein receptor H1 allows cleavage of the internal signal sequence

Human asialoglycoprotein receptor H1 is a single-spanning membrane protein with an amino-terminal domain of 40 residues exposed to the cytoplasm and the carboxyl-terminal domain translocated to the exoplasmic side of the membrane. It has been shown earlier that the transmembrane segment functions as an internal uncleaved signal sequence for insertion into the endoplasmic reticulum. In a deletion protein lacking almost the entire cytoplasmic domain, the signal sequence is cleaved at the carboxyl-terminal end of the transmembrane segment. All available criteria suggest that the protein is processed by signal peptidase. The cytoplasmic domain of the receptor does not directly inhibit signal cleavage since it does not detectably hinder cleavage of the normally amino-terminal signal sequence of influenza hemagglutinin in fusion proteins. We suggest that by its size or structure it affects the position of the receptor in the membrane and thus the accessibility of the potential cleavage site to signal peptidase.     

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.     

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.     

Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT

I gamma CAT is a hybrid protein that inserts into the membrane of the endoplasmic reticulum as a type II membrane protein. These proteins span the membrane once and expose the NH2-terminal end on the cytoplasmic side and the COOH terminus on the exoplasmic side. I gamma CAT has a single hydrophobic segment of 30 amino acid residues that functions as a signal for membrane insertion and anchoring. The signal-anchor region in I gamma CAT was analyzed by deletion mutagenesis from its COOH-terminal end (delta C mutants). The results show that the 13 amino acid residues on the amino-terminal side of the hydrophobic segment are not sufficient for membrane insertion and translocation. Mutant proteins with at least 16 of the hydrophobic residues are inserted into the membrane, glycosylated, and partially proteolytically processed by a microsomal protease (signal peptidase). The degree of processing varies between different delta C mutants. Mutant proteins retaining 20 or more of the hydrophobic amino acid residues can span the membrane like the parent I gamma CAT protein and are not proteolytically processed. Our data suggest that in the type II membrane protein I gamma CAT, the signals for membrane insertion and anchoring are overlapping and that hydrophilic amino acid residues at the COOH-terminal end of the hydrophobic segment can influence cleavage by signal peptidase. From this and previous work, we conclude that the function of the signal-anchor sequence in I gamma CAT is determined by three segments: a positively charged NH2 terminus, a hydrophobic core of at least 16 amino acid residues, and the COOH-terminal flanking hydrophilic segment.     

The carboxyl-terminal valine is required for transport of glycoprotein CD8 alpha from the endoplasmic reticulum to the intermediate compartment

There is evidence that a carboxyl-terminal valine residue is an anterograde transport signal for type I transmembrane proteins. Removal of the signal would either delay glycosylation in the Golgi complex of proteins destined to recycle to the endoplasmic reticulum or determine accumulation in the endoplasmic reticulum of newly synthesized proteins destined for the plasma membrane. We used the human CD8 alpha glycoprotein to investigate the role of the carboxyl-terminal valine in the exocytic pathway. Using immunofluorescence light microscopy, metabolic labeling, and cell fractionation, we demonstrate that removal of the carboxyl-terminal valine residue delays transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment. Removal of the residue did not affect the other steps of the exocytic pathway or the folding/dimerization and glycosylation processes. Therefore, it is likely that this signal plays a role in the transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment either before or during the formation of the transport vesicles that drive the exit the protein from the endoplasmic reticulum.     

Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean

Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity.     

Switching of the homooligomeric ATP-binding cassette transport complex MDL1 from post-translational mitochondrial import to endoplasmic reticulum insertion

The ATP-binding cassette transporter MDL1 of Saccharomyces cerevisiae has been implicated in mitochondrial quality control, exporting degradation products of misassembled respiratory chain complexes. In the present study, we identified an unusually long leader sequence of 59 amino acids, which targets MDL1 to the inner mitochondrial membrane with its nucleotide-binding domain oriented to the matrix. By contrast, MDL1 lacking this leader sequence is directed into the endoplasmic reticulum membrane with the nucleotide-binding domain facing the cytosol. Remarkably, in both targeting routes, the ATP-binding cassette transporter maintains its intrinsic properties of membrane insertion and assembly, leading to homooligomeric complexes with similar activities in ATP hydrolysis. The physiological consequences of both targeting routes were elucidated in cells lacking the mitochondrial ATP-binding cassette transporter ATM1, which is essential for biogenesis of cytosolic iron-sulfur proteins. The mitochondrial MDL1 complex can complement ATM1 function, whereas the endoplasmic reticulum-targeted version, as well as MDL1 mutants deficient in ATP binding and hydrolysis, cannot overcome the Deltaatm1 growth phenotype.     

The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum

Signal sequences for insertion of proteins into the endoplasmic reticulum induce translocation of either the C- or the N-terminal sequence across the membrane. The end that is translocated is primarily determined by the flanking charges and the hydrophobic domain of the signal. To characterize the hydrophobic contribution to topogenesis, we have challenged the translocation machinery in vivo in transfected COS cells with model proteins differing exclusively in the apolar segment of the signal. Homo-oligomers of hydrophobic amino acids as different in size and shape as Val(19), Trp(19), and Tyr(22) generated functional signal sequences with similar topologies in the membrane. The longer a homo-oligomeric sequence of a given residue, the more N-terminal translocation was obtained. To determine the topogenic contribution of all uncharged amino acids in the context of a hydrophobic signal sequence, two residues in a generic oligoleucine signal were exchanged for all uncharged amino acids. The resulting scale resembles a hydrophobicity scale with the more hydrophobic residues promoting N-terminal translocation. In addition, the helix breakers glycine and proline showed a position-dependent effect, which raises the possibility of a conformational contribution to topogenesis.     

Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages

The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the α-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain.     

Efficient glycosylphosphatidylinositol (GPI) modification of membrane proteins requires a C-terminal anchoring signal of marginal hydrophobicity

Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is "marginal": less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.     

Targeting and membrane-insertion of a sunflower oleosin in vitro and in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: the central hydrophobic domain contains more than one signal sequence,and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism

A range of N- and C-terminal deletions of an oleosin from Helianthus annuus L. were used to study the endoplasmic reticulum (ER) targeting and membrane insertion of this protein both in vitro and in vivo in yeast ( Saccharomyces cerevisiae). Neither the N- nor the C-terminal hydrophilic domains are important for targeting and/or membrane insertion, with all the information required for these processes located within the central hydrophobic region of the protein. However, in vitro membrane-insertion experiments suggest that these domains are important for a correct topology of the oleosin within the ER membrane. The first half of the hydrophobic central domain, flanked by the positively charged N-terminal domain, is likely to function as a type-II signal-anchor (SAII) sequence. However, in the absence of the N-terminal 26 residues of this domain, the proline-knot region and the second half of this hydrophobic domain are sufficient to direct oleosin to the ER and to allow stable (but far less efficient) integration of the protein into the membrane. Taken together, these results indicate that oleosin contains more than one domain that is capable of interacting with the signal recognition particle to direct the protein to the ER membrane.