NHE3 contains a cleavable signal peptide followed by 11 membrane spanning domains: New insights into topology

Abstract Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git−) defective in the uptake and metabolism of GroPIns. One mutant was found to be affected in the gene encoding the transcription factor, SPT7. Mutants of the positive regulatory gene INO2, but not of its partner, INO4, also have the Git− phenotype. Another mutant was complemented by a single open reading frame (ORF) termed GIT1 (glycerophosphoinositol). This ORF consists of 1556 bp predicted to encode a polypeptide of 518 amino acids and 57.3 kD. The predicted Git1p has similarity to a variety of S. cerevisiae transporters, including a phosphate transporter (Pho84p), and both inositol transporters (Itr1p and Itr2p). Furthermore, Git1p contains a sugar transport motif and 12 potential membrane-spanning domains. Transport assays performed on a git1 mutant together with the above evidence indicate that the GIT1 gene encodes a permease involved in the uptake of GroPIns.

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Intramembrane proteolysis and post-targeting functions of signal peptides

Biochemical Society Transactions ◽

10.1042/bst0311243 ◽

2003 ◽

Vol 31 (6) ◽

pp. 1243-1247 ◽

Cited By ~ 34

Author(s):

B. Martoglio

Keyword(s):

Endoplasmic Reticulum ◽

Lipid Bilayer ◽

Signal Peptide ◽

Eukaryotic Cells ◽

Endoplasmic Reticulum Membrane ◽

Signal Peptides ◽

Peptide Fragments ◽

Intramembrane Proteolysis ◽

Signal Sequences ◽

Membrane Spanning

Signal sequences are the addresses of proteins destined for secretion. In eukaryotic cells, they mediate targeting to the endoplasmic reticulum membrane and insertion into the translocon. Thereafter, signal sequences are cleaved from the pre-protein and liberated into the endoplasmic reticulum membrane. We have recently reported that some liberated signal peptides are further processed by the intramembrane-cleaving aspartic protease signal peptide peptidase. Cleavage in the membrane-spanning portion of the signal peptide promotes the release of signal peptide fragments from the lipid bilayer. Typical processes that include intramembrane proteolysis is the regulatory or signalling function of cleavage products. Likewise, signal peptide fragments liberated upon intramembrane cleavage may promote such post-targeting functions in the cell.

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Structure and expression of human IgG FcRII(CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes.

Journal of Experimental Medicine ◽

10.1084/jem.170.4.1369 ◽

1989 ◽

Vol 170 (4) ◽

pp. 1369-1385 ◽

Cited By ~ 200

Author(s):

D G Brooks ◽

W Q Qiu ◽

A D Luster ◽

J V Ravetch

Keyword(s):

Structural Heterogeneity ◽

Amino Acid Sequences ◽

Cdna Clones ◽

Membrane Glycoproteins ◽

Igg Binding ◽

Genes Expression ◽

Alternatively Spliced ◽

Membrane Spanning ◽

High Degree ◽

Membrane Spanning Domains

The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. These clones predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains. The extracellular domains display sequence homology to other Fc gamma Rs and members of the Ig supergene family. A minimum of three genes (Fc gamma RIIa, IIa', and Fc gamma RIIb) encode these transcripts, which demonstrate highly related extracellular and membrane spanning domains. IIa/IIa' differ substantially in the intracytoplasmic domain from IIb. Alternative splicing of the IIb gene generates further heterogeneity in both NH2- and COOH-terminal domains of the predicted proteins. Comparison to the murine homologues of these molecules reveals a high degree of conservation between the products of one of these genes, Fc gamma RIIb, and the murine beta gene in primary sequence, splicing pattern, and tissue distribution. In contrast, the sequence of IIa' indicates its relationship to the beta-like genes, with mutation giving rise to a novel cytoplasmic domain, while IIa is a chimera of both alpha- and beta-like genes. Expression of these cDNA molecules by transfection results in the appearance of IgG binding molecules that bear the epitopes defined by the FcRII(CD32) mAbs previously described.

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Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor.

The Journal of Cell Biology ◽

10.1083/jcb.120.2.325 ◽

1993 ◽

Vol 120 (2) ◽

pp. 325-338 ◽

Cited By ~ 90

Author(s):

B L Tang ◽

S H Wong ◽

X L Qi ◽

S H Low ◽

W Hong

Keyword(s):

Golgi Apparatus ◽

Brefeldin A ◽

Cell Types ◽

In Vitro Translation ◽

Tubular Structures ◽

Human Sequence ◽

Intermediate Compartment ◽

Membrane Spanning ◽

Membrane Spanning Domains

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.

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LMP-1's Transmembrane Domains Encode Multiple Functions Required for LMP-1's Efficient Signaling

Journal of Virology ◽

10.1128/jvi.76.22.11551-11560.2002 ◽

2002 ◽

Vol 76 (22) ◽

pp. 11551-11560 ◽

Cited By ~ 18

Author(s):

Ajamete Kaykas ◽

Kathleen Worringer ◽

Bill Sugden

Keyword(s):

Protein Interactions ◽

Tumor Necrosis Factor Receptor ◽

Wild Type ◽

Multiple Functions ◽

Barr Virus ◽

Signaling Domain ◽

Epstein Barr ◽

Membrane Spanning ◽

Carboxy Terminal ◽

Membrane Spanning Domains

ABSTRACT The latent membrane protein-1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the proliferation of infected B lymphocytes by signaling through its binding to cellular signaling molecules. It apparently mimics members of the tumor necrosis factor receptor family, in particular, CD40, by binding a similar set of cellular molecules as does CD40. LMP-1 differs dramatically in its structure from CD40. LMP-1 has six membrane-spanning domains as opposed to CD40's one. LMP-1 also differs from CD40 in its apparent independence of a ligand for its signaling. We have examined the role of LMP-1's membrane-spanning domains in its signaling. Their substitution with six membrane-spanning domains from the LMP-2A protein of EBV yields a derivative which neither coimmunoprecipitates with LMP-1 nor signals to increase the activity of NF-κB as does wild-type LMP-1. These observations indicate that LMP-1 has specific sequences in its membrane-spanning domains required for these activities. LMP-1's first and sixth membrane-spanning domains have multiple leucine residues potentially similar to leucine-heptad motifs that can mediate protein-protein interactions in membranes (Gurezka et al., J. Biol. Chem. 274:9265-9270, 1999). Substitution of seven leucines in LMP-1's sixth membrane-spanning domain has no effect on its function, whereas similar substitutions in its first membrane-spanning domain yielded a derivative which aggregates as does wild-type LMP-1 but has only 3% of wild-type's ability to signal through NF-κB. Importantly, this derivative complements a mutant of LMP-1 with wild-type membrane-spanning domains but no carboxy-terminal signaling domain. These findings together indicate that the membrane-spanning domains of LMP-1 contribute multiple functions to its signaling.

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Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5126 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5126-5131 ◽

Cited By ~ 69

Author(s):

J A Escobedo ◽

P J Barr ◽

L T Williams

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Growth Factor Receptor ◽

Platelet Derived Growth Factor ◽

Tyrosine Kinase Activity ◽

Down Regulation ◽

Atp Binding Site ◽

Membrane Spanning ◽

Membrane Spanning Domains

Three types of mutations were introduced into the platelet-derived growth factor (PDGF) receptor to cause a loss of PDGF-stimulated tyrosine kinase activity: (i) a point mutation of the ATP-binding site, (ii) a deletion of the carboxyl-terminal region, and (iii) replacement of the membrane-spanning sequences by analogous transmembrane sequences of other receptors. Transfectants expressing mutated receptors bind, 125I-labeled PDGF with a high affinity but had no PDGF-sensitive tyrosine kinase activity, phosphatidylinositol turnover, increase in the intracellular calcium concentration, change in cellular pH, or stimulation of DNA synthesis. However, PDGF-induced receptor down regulation was normal in the mutant cells. These results indicate that the transmembrane sequence has a specific signal-transducing function other than merely serving as a membrane anchor and that the receptor kinase activity is necessary for most responses to PDGF but is not required for receptor down regulation.

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GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains

The EMBO Journal ◽

10.1093/emboj/18.17.4679 ◽

1999 ◽

Vol 18 (17) ◽

pp. 4679-4688 ◽

Cited By ~ 436

Author(s):

S. Fowler

Keyword(s):

Circadian Clock ◽

Photoperiodic Flowering ◽

Membrane Spanning ◽

Membrane Spanning Domains

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Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5

Biochemical Journal ◽

10.1042/bj20051938 ◽

2006 ◽

Vol 397 (2) ◽

pp. 289-295 ◽

Cited By ~ 23

Author(s):

Ayako Enomoto ◽

Fumio Omae ◽

Masao Miyazaki ◽

Yasunori Kozutsumi ◽

Toshitsugu Yubisui ◽

...

Keyword(s):

Electron Transfer ◽

Erythrocyte Membrane ◽

Cytochrome B5 ◽

Soluble Form ◽

Affinity Column ◽

Vitro Assay ◽

Membrane Spanning ◽

Triton X ◽

Membrane Spanning Domains

Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b5 and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG–Des2, mb5 (the membrane form of cytochrome b5) and bovine erythrocyte membrane. The apparent Km and Vmax of Des2 for the substrate N-octanoylsphinganine were 35 μM and 40 nmol·h−1·mg of protein−1 respectively. The Km of the hydroxylase for mb5 was 0.8 μM. Interestingly, mb5 was not replaced with the soluble form of cytochrome b5, which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b5R (NADH-cytochrome b5 reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains. The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and mb5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of mb5 by b5R.

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SMS1, a high-copy suppressor of the yeast mas6 mutant, encodes an essential inner membrane protein required for mitochondrial protein import.

Molecular Biology of the Cell ◽

10.1091/mbc.5.5.529 ◽

1994 ◽

Vol 5 (5) ◽

pp. 529-538 ◽

Cited By ~ 63

Author(s):

K R Ryan ◽

M M Menold ◽

S Garrett ◽

R E Jensen

Keyword(s):

Membrane Protein ◽

Protein Import ◽

Mitochondrial Protein ◽

Mitochondrial Protein Import ◽

Yeast Saccharomyces Cerevisiae ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Inner Membrane Protein ◽

Membrane Spanning ◽

Membrane Spanning Domains

MAS6 encodes an essential inner membrane protein required for mitochondrial protein import in the yeast Saccharomyces cerevisiae (Emtage and Jensen, 1993). To identify new inner membrane import components, we isolated a high-copy suppressor (SMS1) of the mas6-1 mutant. SMS1 encodes a 16.5-kDa protein that contains several potential membrane-spanning domains. The Sms1 protein is homologous to the carboxyl-terminal domain of the Mas6 protein. Like Mas6p, Sms1p is located in the mitochondrial inner membrane and is an essential protein. Depletion of Sms1p from cells causes defects in the import of several mitochondrial precursor proteins, suggesting that Sms1p is a new inner membrane import component. Our observations raise the possibility that Sms1p and Mas6p act together to translocate proteins across the inner membrane.

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