Identification and Characterization of Golgin-84, a Novel Golgi Integral Membrane Protein with a Cytoplasmic Coiled-coil Domain

doi:10.1074/jbc.274.5.2953

Identification and Characterization of Golgin-84, a Novel Golgi Integral Membrane Protein with a Cytoplasmic Coiled-coil Domain*

From the Genetic Disease Research Branch, NHGRI, National Institutes of Health, Bethesda, Maryland 20892

Abstract

The cytoplasmic face of the Golgi contains a variety of proteins with coiled-coil domains. We identified one such protein in a yeast two-hybrid screen, using as bait the peripheral Golgi phosphatidylinositol(4,5)P₂ 5-phosphatase OCRL1 that is implicated in a human disease, the oculocerebrorenal syndrome. The ∼2.8-kilobase mRNA is ubiquitously expressed and abundant in testis; it encodes a 731-amino acid protein with a predicted mass of 83 kDa. Antibodies against the sequence detect a novel ∼84-kDa Golgi protein we termed golgin-84. Golgin-84 is an integral membrane protein with a single transmembrane domain close to its C terminus. In vitro, the protein inserts post-translationally into microsomal membranes with an N-cytoplasmic and C-lumen orientation. Cross-linking indicates that golgin-84 forms dimers, consistent with the prediction of an ∼400-residue dimerizing coiled-coil domain in its N terminus. The dimerization potential is supported by a data base search that showed that the N-terminal 497 residues of golgin-84 contain a coiled-coil domain that when fused to the RET tyrosine kinase domain had the ability to activate it, forming the RET-IIoncogene. Data base searching also indicates golgin-84 is similar in structure and sequence to giantin, a membrane protein that tethers coatamer complex I vesicles to the Golgi.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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) AF085199.
↵‡ To whom correspondence should be addressed: GDRB/NHGRI, 49 Convent Dr., Bethesda, MD 20892-4472; Tel.: 301-402-2039; Fax: 301-402-2170; E-mail: rlnuss{at}nhgri.nih.gov.
↵2 Additional analyses, including data base searching, were performed at various World Wide Web sites, listed athttp://www-biol.univ-mrs.fr/english/logligne.html.
↵3 R. A. Bascom, S. Srinivasan, and R. L. Nussbaum, unpublished results.
Abbreviations:

COPI and COPII

coatamer complex I and II, respectively

PBS

phosphate-buffered saline

DTSSP

dithiobis(sulfosuccinimidylproprionate)

UTR

untranslated region of mRNA

NRK

normal rat kidney

BFA

brefeldin A

ER

endoplasmic reticulum

EGFP

enhanced green fluorescent protein

TMD

transmembrane domain

PAGE

polyacrylamide gel electrophoresis

kb

kilobase(s)

kbp

kilobase pair(s)

bp

base pair(s).
- Received September 24, 1998.
- Revision received November 12, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.