Na+/H+Exchanger NHE3 Has 11 Membrane Spanning Domains and a Cleaved Signal Peptide:  Topology Analysis Using In Vitro Transcription/Translation†

Biochemistry ◽  
2000 ◽  
Vol 39 (27) ◽  
pp. 8102-8112 ◽  
Author(s):  
Mirza Zizak ◽  
Megan E. Cavet ◽  
Denis Bayle ◽  
Chung-Ming Tse ◽  
Stefan Hallen ◽  
...  
Keyword(s):  
Signal Peptide ◽  
In Vitro Transcription ◽  
Topology Analysis ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

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

2000 ◽  
Vol 118 (4) ◽  
pp. A871
Author(s):  
Mirza Zizak ◽  
M E Cavet ◽  
D. Bayle ◽  
C M Tse ◽  
S. Hallen ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

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

1993 ◽  
Vol 120 (2) ◽  
pp. 325-338 ◽  
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.

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

2006 ◽  
Vol 397 (2) ◽  
pp. 289-295 ◽  
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.

Mammalian multidrug-resistance proteins (MRPs)

2011 ◽  
Vol 50 ◽  
pp. 179-207 ◽  
Author(s):  
Andrew J. Slot ◽  
Steven V. Molinski ◽  
Susan P.C. Cole
Keyword(s):  
Multidrug Resistance ◽  
Gene Knockout ◽  
Multidrug Resistance Proteins ◽  
Resistance Proteins ◽  
Gene Knockout Mice ◽  
Membrane Spanning ◽  
Endogenous Metabolites ◽  
Membrane Spanning Domains

Subfamily C of the human ABC (ATP-binding cassette) superfamily contains nine proteins that are often referred to as the MRPs (multidrug-resistance proteins). The ‘short’ MRP/ABCC transporters (MRP4, MRP5, MRP8 and ABCC12) have a typical ABC structure with four domains comprising two membrane-spanning domains (MSD1 and MSD2) each followed by a nucleotide-binding domain (NBD1 and NBD2). The ‘long’ MRP/ABCCs (MRP1, MRP2, MRP3, ABCC6 and MRP7) have five domains with the extra domain, MSD0, at the N-terminus. The proteins encoded by the ABCC6 and ABCC12 genes are not known to transport drugs and are therefore referred to as ABCC6 and ABCC12 (rather than MRP6 and MRP9) respectively. A large number of molecules are transported across the plasma membrane by the MRPs. Many are organic anions derived from exogenous sources such as conjugated drug metabolites. Others are endogenous metabolites such as the cysteinyl leukotrienes and prostaglandins which have important signalling functions in the cell. Some MRPs share a degree of overlap in substrate specificity (at least in vitro), but differences in transport kinetics are often substantial. In some cases, the in vivo substrates for some MRPs have been discovered aided by studies in gene-knockout mice. However, the molecules that are transported in vivo by others, including MRP5, MRP7, ABCC6 and ABCC12, still remain unknown. Important differences in the tissue distribution of the MRPs and their membrane localization (apical in contrast with basolateral) in polarized cells also exist. Together, these differences are responsible for the unique pharmacological and physiological functions of each of the nine ABCC transporters known as the MRPs.

Splicing of a retained intron within ROMK K+ channel RNA generates a novel set of isoforms in rat kidney

1999 ◽  
Vol 276 (3) ◽  
pp. C585-C592 ◽  
Author(s):  
A. H. Beesley ◽  
B. Ortega ◽  
S. J. White
Keyword(s):  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
K Channel ◽  
Major Pathway ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Membrane Spanning Domains ◽  
Darby Canine Kidney

The renal outer medulla K+ channel (ROMK) family of K+ channels may constitute a major pathway for K+ secretion in the distal nephron. To date, four main isoforms of this gene have been identified in the rat that differ only in their NH2-terminal amino acids and that share a common “core exon” that determines the remaining protein sequence. Using RT-PCR, we have identified a new set of ROMK isoforms in rat kidney that are generated by the deletion of a region within the ROMK core sequence that is identifiable as a typical mammalian intron. This splicing event was shown to be reproducible in vitro by detection of deleted ROMK mRNA in Madin-Darby canine kidney (MDCK) cells stably transfected with the gene for ROMK2. Translation of the deletion variant of ROMK2 was confirmed in vitro and visualized in MDCK cells following transient transfection with an enhanced green fluorescent protein tag. The deletion in this core region is predicted to generate hydrophilic proteins that are approximately one-third of the size of native ROMK and lack membrane-spanning domains.

In vitro transcription and translation of simian rotavirus SA11 gene products.

1980 ◽  
Vol 33 (3) ◽  
pp. 1111-1121 ◽  
Author(s):  
B B Mason ◽  
D Y Graham ◽  
M K Estes
Keyword(s):  
In Vitro Transcription ◽  
Gene Products
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