Supplementary data 1-2
FRAP
experiments
on
ARL1-YFP
(Supplementary data
1)
and
YFP-GRIP
(Supplementary data 2) were performed on Golgi bodies in tobacco leaf epidermal cells
transiently transformed with either marker and treated with latrunculin B to stop Golgi
movement (Brandizzi et al. 2002). Timeframe of the experiment is indicated at the right
hand corner in both supplementary videos in seconds. Photobleached Golgi stacks are
indicated by and arrowhead. Photobleaching protocol was carried out as described in
Brandizzi et al. (2002).
Reference
Brandizzi F, Snapp EL, Roberts AG, Lippincott-Schwartz J, Hawes C: Membrane protein
transport between the endoplasmic reticulum and the Golgi in tobacco leaves is energy
dependent but cytoskeleton independent: evidence from selective photobleaching. Plant
Cell 14: 1293-309 (2002).
Supplementary data 3
To investigate whether wild-type ARL1 could have a different effect on the distribution
of GRIP on the Golgi apparatus in comparison to the active ARL1 mutant, we
coexpressed wild-type ARL1CFP and ARL1GTP-CFP with YFP-GRIP (Supplementary
figure 3). We verified that the distribution of YFP-GRIP was similar in the presence of
wild-type and GTP restricted ARL1. Interestingly, we have verified that in the presence
of wild-type ARL1 or its mutant, the cytosolic distribution of YFP-GRIP appeared to be
reduced and the protein was mostly localized on the Golgi apparatus (compare Figure 2B
and Supplementary figure 3). This adds to the suggestion that ARL1 is involved in
recruiting GRIP onto the Golgi apparatus, as heterologous expression of ARL1 would
increase the number of available GRIP-receptors on the Golgi membranes.
It is also interesting that we have not verified a different level of recruitment of GRIP
onto the Golgi in the presence of wild-type ARL1 and active ARL1 mutant. It is possible
that these differences may be not appreciable with the imaging technique in use. An
alternative explanation could be that active and wild-type ARL1 influence the
recruitment of GRIP with different effects. Active ARL1 may increase the residency time
of GRIP onto the Golgi apparatus rather than its amount in comparison to wild-type
1
ARL1, as it may hydrolyze GTP at a lower rate than the wild-type protein. Therefore,
cycles of binding and release of GRIP on the Golgi membranes may be slower in the
presence of active ARL1 in comparison to wild-type ARL1. However, in conditions of
heterologous expression of ARL1 proteins, binding sites of ARL1 proteins to the Golgi
are most likely saturated. A saturable Golgi-binding of ARL1 has also been verified in
mammalian cells (Lu et al., 2001). In consequence, GRIP-binding to the Golgi apparatus
appears similar in the presence of wild-type and ARL1-GTP mutant. Analogously, it has
been shown that active ARF1 mutant reduces the cycling of coatomer on and off Golgi
membranes in comparison to wild-type ARF1 and that there is no difference in the
distribution of coatomer on the Golgi apparatus in the presence of wild-type or ARF1GTP (Presley et al., 2002).
References
Lu L, Horstmann H, Ng C, Hong W: Regulation of Golgi structure and function by ARFlike protein 1 (Arl1). J Cell Sci 114: 4543-55 (2001).
Presley JF, Ward TH, Pfeifer AC, Siggia ED, Phair RD, Lippincott-Schwartz J:
Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport.
Nature 417: 187-93 (2002).
Supplementary figure 3 Legend.
Confocal images of tobacco leaf epidermal cells coexpressing wild-type YFP-GRIP with
either wild-type ARL1-CFP (WT) or ARL1GTP-CFP (GTP). Scale bars = 5μm.
Supplementary data 4
To analyze whether AtGRIP dimerizes in vivo, we expressed it as a YFP fusion (YFPAtGRIP) in tobacco leaf epidermal cells. The cDNA encoding full-length AtGRIP was
obtained as an ABRC clone and was subcloned in pVKH18-En6 upstream a YFP using
BamHI and SacI sites. Prior to biochemical analyses we verified the subcellular
localization of the protein. The fluorescent fusion (A) was found to localize to the Golgi
apparatus as shown by colocalization experiments with the Golgi marker ERD2-GFP (B)
and in the cytosol similarly to YFP-GRIP. C = merged image of A and B; scale bar = 5
μm.
2
Leaf segments expressing YFP-AtGRIP were subjected to protein extraction in NE buffer
without dithiothreitol (dTT). The resulting extract was then divided into two portions, one
of which was combined with 2x native sample buffer (1 M sucrose, 20 mM Tris pH 8.8, 5
mM EDTA, 0.1% (w/v) bromophenol blue) and loaded on a native acrylamide gel. The
other portion was combined with sample buffer plus 2.5% (w/v) SDS and 2 mM dTT and
boiled for 5 minutes before loading on the native acrylamide gel. Native PAGE was
followed by western blotting with anti-GFP serum. As shown in Supplementary data 4D,
in the denatured sample two bands were visible; the lower of these corresponds to the
predicted molecular weight of monomeric YFP-AtGRIP fusion (117 kDa; full
arrowhead), while the upper is of similar molecular weight to the band in the native
sample. We conclude that the larger band present in both samples represents dimeric
YFP-AtGRIP. Its presence in the denatured sample suggests a strong interaction between
the two monomers, as otherwise only the lower band would be detected in the denatured
sample. These data indicate that AtGRIP is capable of dimerization in planta and validate
the molecular modeling of AtGRIP.
3