Molecular Cloning and Expression of a 58-kDa cis-Golgi and Intermediate Compartment Protein (*)
- From the (1)Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-17177 Stockholm, Sweden and Uppsala Branch, Box 595, S-75124 Uppsala, Sweden, and the
- (2)Department of Biochemistry and Molecular Biology, University of Bergen, Årstadveien 19, N-5009 Bergen, Norway
- § To whom correspondence should be addressed: Ludwig Institute for Cancer Research, Box 240, S-17177 Stockholm, Sweden. Tel.: 46-8-7287103; Fax: 46-8-332812.
Abstract
An abundant 58-kDa (p58) homodimeric and hexameric microsomal membrane protein has been biochemically characterized and localized to tubulo-vesicular elements at the endoplasmic reticulum-Golgi interface and the cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllöf, B., and Saraste, J.(1992) J. Cell Sci. 103, 321-333). Here we report the purification of p58 by two-dimensional gel electrophoresis, and the cloning and sequencing of the rat and part of the Xenopus laevis cDNAs. The rat cDNA encodes a 517-amino acid protein having a putative signal sequence, a transmembrane domain close to the C terminus and a short cytoplasmic tail. The C-terminal tail contains a double-lysine motif (KKFF), known to mediate retrieval of proteins from the Golgi back to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identified human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpression of c-Myc-tagged p58 resulted in accumulation of the protein both in the endoplasmic reticulum and in an apparently enlarged Golgi complex, as well as its leakage to the plasma membrane. Immunolocalization using antibodies raised against a lumenal peptide stained the total cellular pool of p58, while anti-tail peptide antibodies detected p58 only in a restricted Golgi region. This suggests that the C-terminal tail of p58 located in the endoplasmic reticulum and transport intermediates is hidden, but becomes exposed when the protein reaches the Golgi complex.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(™)/EMBL Data Bank with accession number(s) U44129 (rat) and U44130 (X. laevis).
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↵1 The abbreviations used are:
- ER
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endoplasmic reticulum
- PAGE
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polyacrylamide gel electrophoresis
- SPDP
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N-succinimidyl 3-(2-pyridyldithio)-propionate
- FITC
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fluorescein isothiocyanate
- TRITC
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tetramethylrhodamine B isothiocyanate
- BHK
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baby hamster kidney
- PCR
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polymerase chain reaction.
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- Received September 11, 1995.
- Revision received December 18, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
