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Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Rojo M, Pepperkok R, Emery G, Kellner R, Stang E, Parton RG, Gruenberg J - J. Cell Biol. (1997)

Bottom Line: Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment.Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules.These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

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p23 is a member of the p24 family of proteins. (A)  cDNA-derived amino acid sequence of the BHKp23 precursor.  The predicted cleavage site for the signal peptidase is indicated  by an arrowhead. The peptides used for generation of antibodies  are boxed and their names given in italics. Peptides LP1 and LP2  were identified by microsequencing. The consensus site for glycosilation is underlined, the putative transmembrane domain is  typed in bold characters. These sequence data are available from  GenBank/EMBL/DDBJ under accession number AJ001513. (B)  Dendogram of the p24 family. Only those proteins whose full-length sequence is known were analyzed, the accession numbers  are indicated in parenthesis. Alignment and dendogram were  performed with programs of Genetics Computer Group (Madison, WI). From the known yeast proteins mammalian p23 displays the highest similarity to yeast erv25p. (C) Characterization  of antibodies against BHKp23. A membrane fraction enriched  for p23 (interface M2 of Fig. 7) was subjected to 2D gel electrophoresis, blotted onto nitrocellulose, and then stained with PROTOGOLD (protein stain) or with LP1, LP2, or CT. The p23 protein  (arrowhead) was already detected by protein stain and was specifically decorated with all three antibodies. Lines indicate the position of molecular mass markers (200, 116, 97, 66, 45, 31, and 22 kD).
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Figure 1: p23 is a member of the p24 family of proteins. (A) cDNA-derived amino acid sequence of the BHKp23 precursor. The predicted cleavage site for the signal peptidase is indicated by an arrowhead. The peptides used for generation of antibodies are boxed and their names given in italics. Peptides LP1 and LP2 were identified by microsequencing. The consensus site for glycosilation is underlined, the putative transmembrane domain is typed in bold characters. These sequence data are available from GenBank/EMBL/DDBJ under accession number AJ001513. (B) Dendogram of the p24 family. Only those proteins whose full-length sequence is known were analyzed, the accession numbers are indicated in parenthesis. Alignment and dendogram were performed with programs of Genetics Computer Group (Madison, WI). From the known yeast proteins mammalian p23 displays the highest similarity to yeast erv25p. (C) Characterization of antibodies against BHKp23. A membrane fraction enriched for p23 (interface M2 of Fig. 7) was subjected to 2D gel electrophoresis, blotted onto nitrocellulose, and then stained with PROTOGOLD (protein stain) or with LP1, LP2, or CT. The p23 protein (arrowhead) was already detected by protein stain and was specifically decorated with all three antibodies. Lines indicate the position of molecular mass markers (200, 116, 97, 66, 45, 31, and 22 kD).

Mentions: We identified in a light membrane fraction of BHK cells a polypeptide of 23 kD (see below, Fig. 1 C), which was resistant to membrane extraction by carbonate treatment at pH 11, a behavior characteristic for transmembrane proteins. The protein was relatively abundant, as it could be visualized by protein stain in a total cellular extract (not shown). Microsequencing of p23 revealed that two tryptic fragments (Fig. 1 A, LP1 and LP2) corresponded to the sequences of several human ESTs. These ESTs shared sequence homology to emp24p, a yeast protein involved in biosynthetic membrane transport (Schimmöller et al., 1995). Several homologues of emp24p have been identified, including in mammalian cells, and all these form a novel family of transmembrane proteins (Fig. 1 B). We used nucleic acid probes generated from one human EST clone to screen a BHK cDNA library, and obtained several cDNA clones that were identical in sequence. They always coded for both peptide sequences (Fig. 1 A), but differed in length towards the 5′ end. The clones containing a putative initiator methionine within a consensus Kozak sequence (Kozak, 1989) coded for an open reading frame of 219 amino acids (these sequence data are available from GenBank/EMBL/DDBJ under accession number AJ001513). The open reading frame of the isolated cDNA clones was found to be >97% identical at the amino acid level to Tmp21, a protein by then identified in human and rat (Blum et al., 1996: these sequence data are available from GenBank/EMBL/DDBJ under accession numbers X97442 and X97443) and to rabbit p23 (Sohn et al., 1996; GenBank/EMBL/DDBJ under accession number X98303). BHKp23 exhibits significantly more homology to these three proteins (⩾95%) than to any other member of the p24 family (20–30%; Sohn et al., 1996), indicating that it may correspond to the hamster ortholog of human/rat Tmp21 and rabbit p23 (Fig. 1 B).


Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Rojo M, Pepperkok R, Emery G, Kellner R, Stang E, Parton RG, Gruenberg J - J. Cell Biol. (1997)

p23 is a member of the p24 family of proteins. (A)  cDNA-derived amino acid sequence of the BHKp23 precursor.  The predicted cleavage site for the signal peptidase is indicated  by an arrowhead. The peptides used for generation of antibodies  are boxed and their names given in italics. Peptides LP1 and LP2  were identified by microsequencing. The consensus site for glycosilation is underlined, the putative transmembrane domain is  typed in bold characters. These sequence data are available from  GenBank/EMBL/DDBJ under accession number AJ001513. (B)  Dendogram of the p24 family. Only those proteins whose full-length sequence is known were analyzed, the accession numbers  are indicated in parenthesis. Alignment and dendogram were  performed with programs of Genetics Computer Group (Madison, WI). From the known yeast proteins mammalian p23 displays the highest similarity to yeast erv25p. (C) Characterization  of antibodies against BHKp23. A membrane fraction enriched  for p23 (interface M2 of Fig. 7) was subjected to 2D gel electrophoresis, blotted onto nitrocellulose, and then stained with PROTOGOLD (protein stain) or with LP1, LP2, or CT. The p23 protein  (arrowhead) was already detected by protein stain and was specifically decorated with all three antibodies. Lines indicate the position of molecular mass markers (200, 116, 97, 66, 45, 31, and 22 kD).
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Figure 1: p23 is a member of the p24 family of proteins. (A) cDNA-derived amino acid sequence of the BHKp23 precursor. The predicted cleavage site for the signal peptidase is indicated by an arrowhead. The peptides used for generation of antibodies are boxed and their names given in italics. Peptides LP1 and LP2 were identified by microsequencing. The consensus site for glycosilation is underlined, the putative transmembrane domain is typed in bold characters. These sequence data are available from GenBank/EMBL/DDBJ under accession number AJ001513. (B) Dendogram of the p24 family. Only those proteins whose full-length sequence is known were analyzed, the accession numbers are indicated in parenthesis. Alignment and dendogram were performed with programs of Genetics Computer Group (Madison, WI). From the known yeast proteins mammalian p23 displays the highest similarity to yeast erv25p. (C) Characterization of antibodies against BHKp23. A membrane fraction enriched for p23 (interface M2 of Fig. 7) was subjected to 2D gel electrophoresis, blotted onto nitrocellulose, and then stained with PROTOGOLD (protein stain) or with LP1, LP2, or CT. The p23 protein (arrowhead) was already detected by protein stain and was specifically decorated with all three antibodies. Lines indicate the position of molecular mass markers (200, 116, 97, 66, 45, 31, and 22 kD).
Mentions: We identified in a light membrane fraction of BHK cells a polypeptide of 23 kD (see below, Fig. 1 C), which was resistant to membrane extraction by carbonate treatment at pH 11, a behavior characteristic for transmembrane proteins. The protein was relatively abundant, as it could be visualized by protein stain in a total cellular extract (not shown). Microsequencing of p23 revealed that two tryptic fragments (Fig. 1 A, LP1 and LP2) corresponded to the sequences of several human ESTs. These ESTs shared sequence homology to emp24p, a yeast protein involved in biosynthetic membrane transport (Schimmöller et al., 1995). Several homologues of emp24p have been identified, including in mammalian cells, and all these form a novel family of transmembrane proteins (Fig. 1 B). We used nucleic acid probes generated from one human EST clone to screen a BHK cDNA library, and obtained several cDNA clones that were identical in sequence. They always coded for both peptide sequences (Fig. 1 A), but differed in length towards the 5′ end. The clones containing a putative initiator methionine within a consensus Kozak sequence (Kozak, 1989) coded for an open reading frame of 219 amino acids (these sequence data are available from GenBank/EMBL/DDBJ under accession number AJ001513). The open reading frame of the isolated cDNA clones was found to be >97% identical at the amino acid level to Tmp21, a protein by then identified in human and rat (Blum et al., 1996: these sequence data are available from GenBank/EMBL/DDBJ under accession numbers X97442 and X97443) and to rabbit p23 (Sohn et al., 1996; GenBank/EMBL/DDBJ under accession number X98303). BHKp23 exhibits significantly more homology to these three proteins (⩾95%) than to any other member of the p24 family (20–30%; Sohn et al., 1996), indicating that it may correspond to the hamster ortholog of human/rat Tmp21 and rabbit p23 (Fig. 1 B).

Bottom Line: Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment.Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules.These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

Show MeSH
Related in: MedlinePlus