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V R Lingappa

Publications and source records attributed to V R Lingappa.

At least 37 records · Page 2Linked to original sources
PubMed1993

Amino-terminal assembly of human P-glycoprotein at the endoplasmic reticulum is directed by cooperative actions of two internal sequences.

Transmembrane topology of polytopic integral membrane proteins is established during protein synthesis at the endoplasmic reticulum membrane. For some polytopic proteins, sequential and independent signal, stop transfer, and/or signal anchor sequences contained in the nascent chain direct this process. Here we define the topology of human P-glycoprotein (MDR1) through the first two transmembrane regions (TM1 and TM2, respectively) of the amino-terminal half of the protein. We show that unlike TM7 and TM8, which comprise homologous regions in the carboxyl half of the protein (Skach, W., Calayag, M. C., and Lingappa, V. (1993) J. Biol. Chem. 268, 6903-6908), TM1 and TM2 achieve the orientation predicted by conventional structural models. However, TM1 and TM2 appear to utilize a mechanism of biogenesis different in a key respect from that observed in multispanning proteins studied previously. TM1 and TM2, with their flanking regions, independently direct the topology observed for each of these sequences in the native protein. Each can interact with signal recognition particle to direct targetting to the endoplasmic reticulum, nascent chain translocation, and correct transmembrane orientation. Unlike the transmembrane regions of previously studied multispanning membrane proteins, neither TM1 nor TM2 alone is sufficient to integrate the chain into the membrane. However, when TM1 and TM2 are both present, as occurs in native MDR1, integration is achieved. These results suggest that cooperative interactions between TM1 and TM2 are necessary for chain integration and thus add a new complexity to the current view of polytopic integral membrane protein biogenesis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Analysis of a pause transfer sequence from apolipoprotein B.

In contrast to typical secretory proteins, apolipoprotein B pauses at distinct points along the nascent chain during its translocation into the lumen of the endoplasmic reticulum. Specific pause transfer sequences mediate such discrete pauses in the translocation of apolipoprotein B. These sequences have been shown to confer this translocational behavior to heterologous chimeric proteins. To investigate the function of pause transfer sequences, we: (i) examine whether the multiple pause transfer sequences of apolipoprotein B act independently or are dependent upon the action of upstream pause transfer sequences, (ii) identify residues of the prototypical B' pause transfer sequence that are involved in pausing, and (iii) determine whether the stopping step of a translocational pause is a consequence of translational pausing, as has been suggested by other investigators. We conclude that pause transfer sequences act independently of each other and of translation; translocational pausing occurs even in the absence of ongoing protein synthesis. Furthermore, like other topogenic sequences such as signal sequences, pause transfer sequences are degenerate in structure yet have distinctive features necessary for their action. This characterization of the B' pause transfer sequence may aid in the identification of such sequences elsewhere in apolipoprotein B and in other proteins and has implications for the mechanism of translocational pausing.

Amino Acid Sequence↗
PubMed1993

Evidence for an alternate model of human P-glycoprotein structure and biogenesis.

We have studied the transmembrane topology of human P-glycoprotein (MDR1) using protein chimeras in Xenopus oocytes and full-length native protein in a cell-free translation system. We find both in vivo and in vitro, that the peptide region between putative transmembrane helices (TM) 8 and 9 resides in the endoplasmic reticulum lumen not in the cytosol as predicted. The topology of the carboxyl-terminal half of MDR1 therefore appears distinct from the homologous amino-terminal half in which the corresponding region between TM2 and TM3 is cytosolic. Thus, topogenic sequences encoded in the homologous amino and carboxyl domains of MDR1 direct fundamentally different events in biogenesis of the two halves of MDR1. We conclude that the transmembrane topology of MDR1, an important member of the ATP binding cassette (ABC) transporter superfamily, is not as predicted and should be revised.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Pause transfer: a topogenic sequence in apolipoprotein B mediates stopping and restarting of translocation.

Previously, we described the stepwise translocation of a large amino-terminal fragment of apolipoprotein B (apo B15) in which the nascent secretory chain translocates through a series of distinct, nonintegrated transmembrane intermediates with large domains exposed to the cytoplasm. Thus, apo B15 appears to stop and restart translocation at several points. We have identified a sequence of amino acids in apo B15 that confers this behavior on a heterologous chimeric protein. In addition, we dissect pausing into two distinct steps, stopping and restarting, thereby trapping otherwise transient intermediates. Finally, we demonstrate the function of a second "pause transfer" sequence over 200 amino acids downstream in apo B15 that restarts translocation posttranslationally, suggesting that multiple pause transfer sequences are involved in the biogenesis of apolipoprotein B.

Amino Acid Sequence↗
PubMed1991

Expression of human recombinant lipocortin I in a wheat-germ cell-free system and Xenopus oocytes. Lipocortin is not secreted.

Lipocortin I has been presumed to be synthesized and secreted in response to glucocorticoids yet the amino acid sequence of lipocortin I reveals no signal sequence typically necessary for proteins to enter the secretory pathway. The translocation of lipocortin I across membranes was analyzed in a cell-free system and in Xenopus oocytes. Based on the published sequence, the cDNA of human lipocortin I was cloned and expressed in Escherichia coli. Lipocortin I was purified and used to raise monoclonal antibodies. To test whether lipocortin I is secreted in vitro, transcribed lipocortin mRNA was translated in a wheat germ cell-free system in the absence and presence of microsomal membranes. Prolactin mRNA was used as a control for translocation of newly synthesized protein into membrane vesicles. Prolactin, but not lipocortin I, was translocated into the membranes. To test for secretion of lipocortin I in vivo, Xenopus oocytes were co-injected with transcripts encoding lipocortin I and prolactin, with and without the signal sequence. Prolactin with the signal sequence was released into the medium. However, neither prolactin without a signal sequence nor lipocortin I was released. Carbonate extraction, using an integral transmembrane protein as control, revealed no evidence for membrane integration of lipocortin I. Thus lipocortin I is not a secreted protein.

Animals↗
PubMed1990

New variation on the translocation of proteins during early biogenesis of apolipoprotein B.

Apolipoprotein B (apo B) is crucial for the transport of cholesterol in humans. It is a large secretory protein that mediates the uptake of low-density lipoproteins and renders several forms of lipid droplets soluble in the blood. The binding of lipid by apo B also prevents this hydrophobic protein from precipitating in aqueous solution. In the endoplasmic reticulum, nascent secretory proteins must be translocated through an aqueous channel in the membrane into the aqueous lumen, so some novel form of processing may be necessary to maintain the solubility of apo B during its translocation. We have discovered that the biogenesis of apo B in cell-free systems does indeed involve a new variation on protein translocation: unlike typical secretory proteins, apo B is synthesized as a series of transmembrane chains with large cytoplasmic domains and progressively longer amino-terminal regions that are protected against added proteases during the translocation process. In contrast to typical transmembrane proteins, these transmembrane chains are not integrated into the bilayer. Moreover, the transmembrane chains with the shortest protected domains are precursors of forms whose protection is progressively extended to cover the length of the protein. This stepwise conversion occurs post-translationally for the most part. We propose a model on the basis of these findings for the biogenesis of apo B.

Animals↗
PubMed1990

Unusual topogenic sequence directs prion protein biogenesis.

Biosynthetic studies of the prion protein (PrP) have shown that two forms of different topology can be generated from the same pool of nascent chains in cell-free translation systems supplemented with microsomal membranes. A transmembrane form is the predominant product generated in wheat germ (WG) extracts, whereas a completely translocated (secretory) form is the major product synthesized in rabbit reticulocyte lysates (RRL). An unusual topogenic sequence within PrP is now shown to direct this system-dependent difference. The actions of this topogenic sequence were independent of on-going translation and could be conferred to heterologous proteins by the engineering of a discrete set of codons. System-dependent topology conferred by addition of RRL to WG translation products suggests that this sequence interacts with one or more cytosolic factors.

Animals↗
PubMed1990

Non-hydrophobic extracytoplasmic determinant of stop transfer in the prion protein.

A universal feature of integral transmembrane proteins is a hydrophobic peptide segment that spans the lipid bilayer. These hydrophobic domains are important for terminating the translocation of the polypeptide chain across the membrane of the endoplasmic reticulum (a process termed stop transfer) and for integrating the protein into the bilayer. But a role for extracytoplasmic sequences in stop transfer and transmembrane integration has not previously been shown. Recently, a sequence which directs an unusual mode of stop transfer has been identified in the prion protein. This brain glycoprotein exists in two isoforms, which are identical both in primary amino-acid sequence and in containing phosphatidylinositol glycolipid linkages at their C termini, which can be cleaved by a phosphatidylinositol-specific phospholipase C9. But only one of the isoforms (PrPC) is released from cells on treatment with this phospholipase, indicating that the two isoforms have either different subcellular locations or transmembrane orientations. Consistent with this is the observation of two different topological forms in cell-free systems. An unusual topogenic sequence in the prion protein seems to direct these alternative topologies (manuscript in preparation). In the wheat-germ translation system, this sequence directs nascent chains to a transmembrane orientation; by contrast, in the rabbit reticulocyte lysate system, this sequence fails to cause stop transfer of most nascent chains. We have now investigated determinants in this unusual topogenic sequence that direct transmembrane topology, and have demonstrated that (1) a luminally disposed charged domain is required for stop transfer at the adjacent hydrophobic domain, (2) a precise spatial relationship between these domains is essential for efficient stop transfer, and (3) codons encompassing this hydrophilic extracytoplasmic domain confer transmembrane topology to a heterologous protein when engineered adjacent to the codons for a normally translocated hydrophobic domain. These results identify an unexpected functional domain for stop transfer in the prion protein and have implications for the mechanism of membrane protein biogenesis.

Amino Acid Sequence↗
PubMed1990

The N-terminal (pre-S2) domain of a hepatitis B virus surface glycoprotein is translocated across membranes by downstream signal sequences.

The coding region for the hepatitis B virus surface antigens contains three in-phase ATG codons which direct the synthesis of three related polypeptides. The 24-kilodalton major surface (or S) glycoprotein is initiated at the most distal ATG and is a transmembrane protein whose translocation across the bilayer is mediated by at least two uncleaved signal sequences. The product of the next upstream ATG is the 31-kilodalton pre-S2 protein, which contains 55 additional amino acids attached to the N terminus of the S protein. This pre-S2-specific domain is translocated into the endoplasmic reticulum. Using a coupled in vitro translation-translocation system, we showed that (i) the pre-S2 domain itself lacks functional signal sequence activity, (ii) its translocation across the endoplasmic reticulum membrane is mediated by downstream signals within the S domain, and (iii) the N-terminal signal sequence of the S protein can translocate upstream protein domains in the absence of other signals. The hepatitis B virus pre-S2 protein is an example of a natural protein which displays upstream domain translocation, a phenomenon whose existence was originally inferred from the behavior of synthetic fusion proteins in vitro.

Cloning, Molecular↗
PubMed1989

Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor.

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.

Endoplasmic Reticulum↗
PubMed1988

Sequences beyond the cleavage site influence signal peptide function.

The earliest events in protein secretion include targeting to and translocation across the endoplasmic reticulum membrane. To dissect the mechanism by which signal sequences mediate translocation in eukaryotes, we are examining the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that the protein domain being translocated can have profound impact on the efficiency of the translocation process. Specifically, deletions in the mature prolactin "passenger" domain, beyond the signal cleavage site, reduce the efficiency of signal function. The effect of these deletions on signal function is observed when this signal sequence is in its normal position, at the amino terminus, and when internalized by the addition of 117 amino acids of chimpanzee alpha-globin. Alterations in the interaction of the deletion mutants with the signal recognition particle and with another component of the translocation system, signal peptidase, were observed. Our results suggest that subtle changes in sequences beyond the signal cleavage site can alter the efficiency of co-translational translocation by affecting various signal-receptor interactions.

Amino Acid Sequence↗
PubMed1988

Construction of defined polytopic integral transmembrane proteins. The role of signal and stop transfer sequence permutations.

Signal and stop transfer sequences are discrete regions within a polypeptide chain able to initiate or terminate translocation of the protein across the membrane of the endoplasmic reticulum. We have investigated the role of these topogenic sequences in the biogenesis of polytopic transmembrane proteins. Plasmids encoding various patterns of well-characterized signal and stop transfer sequences fused to a set of topogenically inert passenger domains were constructed. These molecules were expressed by transcription-translation in a cell-free system or by microinjection of transcripts into Xenopus oocytes. The observed orientation with respect to the membrane was dependent on the order of signal and stop transfer sequences in the coding region. These results were used to test the hypothesis that a protein can achieve polytopic transmembrane orientation using combinations of simple topogenic sequences. We conclude that some (but not all) patterns of signal and stop transfer sequences confer polytopic orientation to proteins across the membrane of the endoplasmic reticulum.

Animals↗
PubMed1988

Developmental regulation of prion protein mRNA in brain.

During development of the hamster brain, synthesis of the cellular isoform of the scrapie prion protein (PrPC) was found to be regulated. Low levels of PrP poly(A)+ mRNA were detectable one day after birth. PrP poly(A)+ mRNA reached maximal levels between 10 and 20 days post-partum; thereafter, no change in its level could be detected at ages up to 13 months. In contrast, myelin basic protein poly(A)+ mRNA was shown to reach maximal levels by 30 days of age and thereafter steadily declined in adult brain. Using monospecific PrP antisera, immunoprecipitable cell-free translation products were detected at low levels two days after birth and progressively increased up to 10 days of age. How the PrP mRNA participates in brain development and its function in scrapie prion infection are being investigated.

Animals↗
PubMed1988

A block to the intracellular transport and assembly of hepatitis B surface antigen polypeptides in Xenopus oocytes.

Hepatitis B surface antigen is the major protein of the virion envelope, and is also independently secreted from infected cells as a subviral particle composed exclusively of HBsAg and host-derived lipid. Similar particles are efficiently assembled and secreted by cultured mammalian cells transfected with the gene for HBsAg. In contrast to such cultured cells, Xenopus oocytes microinjected with HBsAg mRNA secrete less than 5% of newly synthesized HBsAg polypeptides. We have examined the HBsAg biosynthetic intermediates in such oocytes and provide evidence that the impaired secretion of HBsAg is due to a discrete block in the assembly of lipoprotein particles.

Animals↗