Biochem. J. (2005) 388, 835–841 (Printed in Great Britain)
The trans-Golgi network GRIP-domain proteins form α-helical homodimers
Michael R. LUKE, Fiona HOUGHTON, Matthew A. PERUGINI and Paul A. GLEESON1
The Russell Grimwade School of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne,
Melbourne, VIC 3010, Australia
A recently described family of TGN (trans-Golgi network) pro-
teins, all of which contain a GRIP domain targeting sequence,
has been proposed to play a role in membrane transport. On
the basis of the high content of heptad repeats, GRIP domain
have the potential to mediate protein–protein interactions. Four
mammalian GRIP domain proteins have been identified which
are targeted to the TGN through their GRIP domains, namely
p230, golgin-97, GCC88 and GCC185. In the present study, we
proteins to interact. Using a combination of immunoprecipitation
experiments of epitope-tagged GRIP domain proteins, cross-
linking experiments and yeast two-hybrid interactions, we have
established that the GRIP proteins can self-associate to form
homodimers exclusively. Two-hybrid analysis indicated that the
N- and C-terminal fragments of GCC88 can interact with them-
selves but not with each other, suggesting that the GRIP domain
proteins form parallel coiled-coil dimers. Analysis of purified
recombinant golgin-97 by CD spectroscopy indicated a 67% α-
helical structure, consistent with a high content of coiled-coil se-
quences. These results support a model for GRIP domain proteins
as extended rod-like homodimeric molecules. The formation of
homodimers, but not heterodimers, indicates that each of the
four mammalian TGN golgins has the potential to function in-
Key words: dimer, golgin, GRIP domain protein, homodimer,
trans-Golgi network, yeast two-hybrid.
On the basis of their high content of coiled coils, these molecules
are predicted to adopt a rod-like or extended fibrous structure.
Members of the family of coiled-coil proteins specifically located
on the Golgi (termed golgins), such as p115 and GM130, have
been shown to be important as tethering molecules and in the
biogenesis of membranes of the Golgi stack [1–5]. A number of
golgins specifically associated with the TGN (trans-Golgi net-
work) have recently been identified [6,7] based on the presence
of a modestly conserved, 45-residue Golgi targeting sequence
located at the C-terminus, called the GRIP domain [8–10].
The importance of the TGN golgins is highlighted by the
tified in yeast, plants and protozoan parasites [10–12]. Alteration
in the levels of TGN golgins has been shown to result in abnor-
malities in TGN structure and/or a block in membrane transport
pathways, indicating that they may play a role similar to the
golgins of the Golgi stack, as either matrix components and/or in
vesicular tethering [7,13,14].
There are four mammalian GRIP domain proteins which have
been identified and all are localized to the TGN of HeLa cells
. The mammalian TGN golgins include a 230 kDa peripheral
membrane protein (p230, also known as golgin-245), shown to
be specifically associated with buds/vesicles of TGN membranes
[15–18], golgin-97 , GGC88 and GGC185 (GCC stands for
Golgi-localized coiled-coil protein) . Like other members of
the golgin family, the sequence of GRIP domain proteins indi-
cates a high percentage (75–85%) of α-helical heptad repeats,
sequences typical of coiled coils. The prediction of coiled-coil
domains suggests that the TGN golgins probably exist as dimers
or oligomers. An important question is whether such dimers/
oligomers are homo-oligomers or whether different members of
the GRIP family can associate to form hetero-oligomers. This is a
relevant issue in defining whether the TGN golgins can function
independent of each other or whether their activities are closely
linked. In the present study, using a combination of approaches,
of one family member.
Epitope-tagged proteins were detected with the monoclonal
antibody anti-Myc (9E10)  and monoclonal antibody anti-
FLAG (M2) (Sigma, Castle Hill, NSW, Australia). Horseradish
peroxidase-conjugated rabbit anti-mouse Ig and porcine anti-
rabbit Ig were obtained from Dako (Carpinteria, CA, U.S.A.).
Rabbit antibodies to GCC88 have been described in . Sheep
anti-rabbit Ig-FITC, sheep anti-mouse Ig-FITC and sheep anti-
human IgG-FITC were purchased from Silenus Laboratories
(Melbourne, VIC, Australia).
Cell culture and transfection
HeLa and COS cells were maintained as monolayers in
Dulbecco’s modified Eagle’s medium, supplemented with 10%
(v/v)foetalcalfserum,2 mMglutamineand100 µg/mlpenicillin/
streptomycin, in a humidified 37◦C incubator with 10% CO2.
Transient transfections of cells were performed using FuGENETM
transfection reagent (Roche, Castle Hill, NSW, Australia) as
described previously .
Abbreviations used: DTSSP, dithiobis(sulphosuccinimidyl propionate); TGN, trans-Golgi network.
1To whom correspondence should be addressed (email email@example.com).
c ?2005 Biochemical Society
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Received 28 October 2004/4 January 2005; accepted 18 January 2005
Published as BJ Immediate Publication 18 January 2005, DOI 10.1042/ BJ20041810
c ?2005 Biochemical Society