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Proteins from a GST pulldown using the 
-adaptin
ear construct and pig brain cytosol were subjected to SDS PAGE and the gel was
stained with Coomassie blue. Bands were excised, diced into 2-mm pieces, and
subjected to an in-gel digestion overnight using modified trypsin (Promega).
Extracted peptides were dried down in prelubricated tubes and reconstituted
in 5 ml of HPLC grade water. A 0.5 µl aliquot of the sample was mixed
with 0.5 µl of matrix (a saturated solution of 2:5, dihydroxybenzoic acid
in water) and subjected to MALDI mass spectrometry using a Perseptive Biosystems
Voyager Elite XL. All MALDI spectra were externally calibrated by using a standard
peptide mixture. Database searching of peptide masses was performed using MS-FIT
(Protein Prospector, University of California San Francisco). Based on an entry
of 63 peptide masses, for one of the bands (band 4), the database search identified
mouse rabaptin-5 as the highest scoring match with 22/63 (34%), giving a 30%
coverage of this protein, whereas the second hit was rat rabaptin-5 with 20/63
(31%) 26% coverage. No other hits were recorded for this band.
Although the only bands in the GST-
ear pulldown that could be identified definitively by MALDI mass spectrometry
were band 1 (MAP1A) and band 4 (rabaptin-5), possible matches were found between
band 2 and the putative actin binding protein ACF7, between band 6 and Hsp70,
and between band 7 and 160 kD neurofilament triplet M protein. The significance
of these matches is currently being assessed. We have also investigated whether
the GGA1 and GGA2 ear domains bind to either -synergin
or rabaptin-5. Figure S1 a shows a Coomassie blue-stained gel of the pulldowns,
the same gel that is shown in Figure 6 of the paper. Supplemental Figure 1 b
shows a blot of the pulldown labeled with antiĞ-synergin,
and demonstrates that GGA1 and GGA2 also bring down -synergin,
although it is not brought down by GST alone. However, the amount of -synergin
pulled down by GGA1 or GGA2 is less than the amount pulled down by -adaptin,
and in addition, GGA1 and GGA2 selectively bring down the higher molecular weight
form of -synergin. -Synergin
is known to be alternatively spliced (Page et al., 1999), so this result suggests
that different splice variants might preferentially interact with different
ear-like domains. Quantification of
the blots using a phosphorimager shows three to four times as much immunoreactivity
in the -adaptin pulldown as in the GGA1
or GGA2 pulldowns. Figure S1 c shows a blot of the pulldowns probed with a rabaptin-5
antibody, and demonstrates that rabaptin-5 comes down not only with GST-,
but also with GST-GGA2, although not with GST-GGA1 or with GST alone.
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Figure S1 |
We have previously shown that -adaptin
binds directly to -synergin
by probing blots of purified His-tagged -synergin
with GST- ear, followed
by anti-GST (Page et al., 1999). This same approach was used to determine whether
GGA1 and GGA2 also bind directly to -synergin,
and whether the interaction with rabaptin-5 is a direct one. Figure S1 d shows
blots of His-tagged -synergin
(specifically the -adaptin
ear-binding domain; His--s)
and His-tagged rabaptin-5 (His-r-5), together with a His-tagged control (His-con),
probed with either GST alone, GST-
ear, GST-GGA1 ear, or GST-GGA2 ear, followed by anti-GST. The GST-
construct strongly labels His-tagged -synergin,
and it also labels His-tagged rabaptin-5, but not the His-tagged control. Thus,
the interaction between the -adaptin
ear and rabaptin-5 appears to be a direct one. The GGA1 and GGA2 constructs
produced less clear cut results. Both constructs were found to label His-tagged
-synergin on blots,
but much more weakly than the -adaptin
construct. They also both labeled rabaptin-5 on the blots, but again the signal
with the -adaptin construct
was stronger. Thus, although under certain conditions the GGA ears can interact
with -synergin and
rabaptin-5, both -synergin
and rabaptin-5 interact more strongly with the -adaptin
ear, and it is possible that the interactions we detect with the GGA1 and GGA2
constructs may not occur under physiological conditions.
To look for additional binding partners that interact directly with the
and/or GGA ear domains, blots of the GST pulldowns were probed with the three
GST constructs, followed by anti-GST (Figure S2, aĞd, left). Because all the
blots contain GST fusion proteins and have been probed with anti-GST, the major
labeled bands are the fusion proteins themselves, as well as aggregates and
breakdown products of the fusion proteins, all of which can be seen in the blot
probed with GST alone, followed by anti-GST (Figure S2 a). However, additional
bands can be seen in the blots probed with the GST-
ear and GST-GGA ear constructs. Particularly prominent is a band of 56 kD, presumably
the same band that can be seen in the Coomassie blue-stained gel, which is pulled
down by all three fusion proteins and which they all recognize on the blots
(Figure S2, arrowheads). There is also a band of ~160 kD, which may also be
visible in the Coomassie blue-stained gel, that is only labeled on blots by
the GST- ear construct,
although it is pulled down by all three constructs (Figure S2 b, filled circles).
It is possible that this protein binds directly to -adaptin,
but only indirectly to GGA1 and GGA2, or alternatively that only the interaction
with -adaptin can occur
when the protein is partially denatured on a Western blot. Still, other proteins
only come down with the g-adaptin construct and can only be labeled with that
construct, including a band of ~100 kD (Figure S2 b, open circle).
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Figure S2 |
To investigate whether these or any other binding partners might be enriched
in clathrin-coated vesicles, blots of clathrin-coated vesicles, together with
equal protein loadings from a microsomal fraction, were probed with GST-
ear, GST-GGA1 ear, GST-GGA2 ear, or GST alone (Figure S2 aĞd, right). There
are two prominent bands of ~75 and 52 kD, highly enriched in clathrin-coated
vesicles, that can be labeled with the GST-
ear construct, as well as several minor bands. In contrast, neither of the GGA
constructs gives strong labeling of bands enriched in clathrin-coated vesicles,
consistent with the lack of enrichment of the GGAs themselves in clathrin-coated
vesicles. These observations extend the view that the GGA ears interact with
a subset of the ear binding partners.
We thank Marino Zerial (European Molecular Biology Laboratory, Heidelberg, Germany) for the antibodies against rabaptin-5 and the His-tagged rabaptin-5 construct.
References
Page, L.J., P.J. Sowerby, W.W.Y. Lui, and M.S. Robinson. 1999.
-Synergin: an EH domain-containing
protein that interacts with -adaptin.
J. Cell Biol. 146:993Ğ1004. [Abstract]
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