Distinct Biogenesis Mechanisms for the Water Channels MIWC and CHIP28 at the Endoplasmic Reticulum

Lan bo Shi, William Skach, Tonghui Ma, A. S. Verkman

Research output: Contribution to journalArticle

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Abstract

MIWC is a 32 kDa mercurial-insensitive water channel [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500] expressed in kidney collecting duct, brain ependymal cells, airways, and other tissues. We showed recently that the homologous water channel CHIP28 spanned the endoplasmic reticulum (ER) membrane 4 times with N- and C-termini in the cytoplasm [Skach et al., (1994) J. Cell Biol. 125, 803-815]. Hydropathy analysis of MIWC indicated up to eight hydrophobic regions (HRs) comprising potential membrane-spanning domains. To determine MIWC transmembrane topology at the ER, 10 cDNA chimeras were constructed which encoded increasing lengths of MIWC upstream from a reporter epitope (prolactin P-domain) at residues 13, 46, 73, 92, 120, 140, 164, 209, 276, and 297, corresponding to putative polar extramembrane loops in the MIWC sequence. The chimeras were translated cell-free (rabbit reticulocyte lysate + ER-derived microsomes) and in Xenopus oocytes. Peptide chains were labeled with [35S]methionine and immunoprecipitated with a P-domain antibody. Transmembrane topology as determined by protease accessibility of the P-reporter indicated six membrane-spanning domains with bland C-termini in the cytoplasm. The predicted topology was confirmed by demonstrating N-linked glycosylation at native residue N131 and an engineered consensus site at residue 197. Membrane integration of the nascent chain, as assayed by extractability at pH 11.5, occurred after synthesis of the first HR (residues 1-46). Translocation was terminated by a stop transfer sequence in the second HR (residues 32-73) as demonstrated by translation of the heterologous construct, [prolactin signal sequence]-[globin]- [HR2]-P. In contrast to these results for MIWC, CHIP28 spanned the ER membrane 4 times, became integrated after 4 HRs (residues 1 - 107), and required HRs 2-4 (residues 35-139) to terminate translocation. Thus, despite their conserved sequences and similar function, significant differences exist in the early biogenesis of water channels CHIP28 and MIWC.

Original languageEnglish (US)
Pages (from-to)8250-8256
Number of pages7
JournalBiochemistry
Volume34
Issue number26
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Aquaporins
Endoplasmic Reticulum
Membranes
Prolactin
Topology
Cytoplasm
Collecting Kidney Tubules
Globins
Conserved Sequence
Reticulocytes
Glycosylation
Protein Sorting Signals
Xenopus
Microsomes
Methionine
Membrane Potentials
Oocytes
Epitopes
Peptide Hydrolases
Complementary DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Distinct Biogenesis Mechanisms for the Water Channels MIWC and CHIP28 at the Endoplasmic Reticulum. / Shi, Lan bo; Skach, William; Ma, Tonghui; Verkman, A. S.

In: Biochemistry, Vol. 34, No. 26, 1995, p. 8250-8256.

Research output: Contribution to journalArticle

Shi, Lan bo ; Skach, William ; Ma, Tonghui ; Verkman, A. S. / Distinct Biogenesis Mechanisms for the Water Channels MIWC and CHIP28 at the Endoplasmic Reticulum. In: Biochemistry. 1995 ; Vol. 34, No. 26. pp. 8250-8256.
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abstract = "MIWC is a 32 kDa mercurial-insensitive water channel [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500] expressed in kidney collecting duct, brain ependymal cells, airways, and other tissues. We showed recently that the homologous water channel CHIP28 spanned the endoplasmic reticulum (ER) membrane 4 times with N- and C-termini in the cytoplasm [Skach et al., (1994) J. Cell Biol. 125, 803-815]. Hydropathy analysis of MIWC indicated up to eight hydrophobic regions (HRs) comprising potential membrane-spanning domains. To determine MIWC transmembrane topology at the ER, 10 cDNA chimeras were constructed which encoded increasing lengths of MIWC upstream from a reporter epitope (prolactin P-domain) at residues 13, 46, 73, 92, 120, 140, 164, 209, 276, and 297, corresponding to putative polar extramembrane loops in the MIWC sequence. The chimeras were translated cell-free (rabbit reticulocyte lysate + ER-derived microsomes) and in Xenopus oocytes. Peptide chains were labeled with [35S]methionine and immunoprecipitated with a P-domain antibody. Transmembrane topology as determined by protease accessibility of the P-reporter indicated six membrane-spanning domains with bland C-termini in the cytoplasm. The predicted topology was confirmed by demonstrating N-linked glycosylation at native residue N131 and an engineered consensus site at residue 197. Membrane integration of the nascent chain, as assayed by extractability at pH 11.5, occurred after synthesis of the first HR (residues 1-46). Translocation was terminated by a stop transfer sequence in the second HR (residues 32-73) as demonstrated by translation of the heterologous construct, [prolactin signal sequence]-[globin]- [HR2]-P. In contrast to these results for MIWC, CHIP28 spanned the ER membrane 4 times, became integrated after 4 HRs (residues 1 - 107), and required HRs 2-4 (residues 35-139) to terminate translocation. Thus, despite their conserved sequences and similar function, significant differences exist in the early biogenesis of water channels CHIP28 and MIWC.",
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N2 - MIWC is a 32 kDa mercurial-insensitive water channel [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500] expressed in kidney collecting duct, brain ependymal cells, airways, and other tissues. We showed recently that the homologous water channel CHIP28 spanned the endoplasmic reticulum (ER) membrane 4 times with N- and C-termini in the cytoplasm [Skach et al., (1994) J. Cell Biol. 125, 803-815]. Hydropathy analysis of MIWC indicated up to eight hydrophobic regions (HRs) comprising potential membrane-spanning domains. To determine MIWC transmembrane topology at the ER, 10 cDNA chimeras were constructed which encoded increasing lengths of MIWC upstream from a reporter epitope (prolactin P-domain) at residues 13, 46, 73, 92, 120, 140, 164, 209, 276, and 297, corresponding to putative polar extramembrane loops in the MIWC sequence. The chimeras were translated cell-free (rabbit reticulocyte lysate + ER-derived microsomes) and in Xenopus oocytes. Peptide chains were labeled with [35S]methionine and immunoprecipitated with a P-domain antibody. Transmembrane topology as determined by protease accessibility of the P-reporter indicated six membrane-spanning domains with bland C-termini in the cytoplasm. The predicted topology was confirmed by demonstrating N-linked glycosylation at native residue N131 and an engineered consensus site at residue 197. Membrane integration of the nascent chain, as assayed by extractability at pH 11.5, occurred after synthesis of the first HR (residues 1-46). Translocation was terminated by a stop transfer sequence in the second HR (residues 32-73) as demonstrated by translation of the heterologous construct, [prolactin signal sequence]-[globin]- [HR2]-P. In contrast to these results for MIWC, CHIP28 spanned the ER membrane 4 times, became integrated after 4 HRs (residues 1 - 107), and required HRs 2-4 (residues 35-139) to terminate translocation. Thus, despite their conserved sequences and similar function, significant differences exist in the early biogenesis of water channels CHIP28 and MIWC.

AB - MIWC is a 32 kDa mercurial-insensitive water channel [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500] expressed in kidney collecting duct, brain ependymal cells, airways, and other tissues. We showed recently that the homologous water channel CHIP28 spanned the endoplasmic reticulum (ER) membrane 4 times with N- and C-termini in the cytoplasm [Skach et al., (1994) J. Cell Biol. 125, 803-815]. Hydropathy analysis of MIWC indicated up to eight hydrophobic regions (HRs) comprising potential membrane-spanning domains. To determine MIWC transmembrane topology at the ER, 10 cDNA chimeras were constructed which encoded increasing lengths of MIWC upstream from a reporter epitope (prolactin P-domain) at residues 13, 46, 73, 92, 120, 140, 164, 209, 276, and 297, corresponding to putative polar extramembrane loops in the MIWC sequence. The chimeras were translated cell-free (rabbit reticulocyte lysate + ER-derived microsomes) and in Xenopus oocytes. Peptide chains were labeled with [35S]methionine and immunoprecipitated with a P-domain antibody. Transmembrane topology as determined by protease accessibility of the P-reporter indicated six membrane-spanning domains with bland C-termini in the cytoplasm. The predicted topology was confirmed by demonstrating N-linked glycosylation at native residue N131 and an engineered consensus site at residue 197. Membrane integration of the nascent chain, as assayed by extractability at pH 11.5, occurred after synthesis of the first HR (residues 1-46). Translocation was terminated by a stop transfer sequence in the second HR (residues 32-73) as demonstrated by translation of the heterologous construct, [prolactin signal sequence]-[globin]- [HR2]-P. In contrast to these results for MIWC, CHIP28 spanned the ER membrane 4 times, became integrated after 4 HRs (residues 1 - 107), and required HRs 2-4 (residues 35-139) to terminate translocation. Thus, despite their conserved sequences and similar function, significant differences exist in the early biogenesis of water channels CHIP28 and MIWC.

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