Sequence of C-terminal tail regions of myosin heavy chains in class XI of Nicotiana benthamiana (Nb

Transcription

1 Fig. S1 Sequence of C-terminal tail regions of myosin heavy chains in class XI of Nicotiana benthamiana (Nb myosin XI-2, -F and K) and BY-2 cell (Nt 170-kD myosin and Nt 175-kD myosin). Amino acids identical between 3 to 5 myosins are shown with a black background. Red and blue double arrows indicate the polypeptide for raising the anti-bm175 peptide and anti-bm175 tail antibody, respectively. The red underline and the sequence in the red brackets indicates the C-terminus portion of dilute domain and putative Rab GTPase (AtRab) binding region (Hashimoto et al., 2008), respectively.

2 Fig. S2 Isolation protocol for the S12 fraction and GFP-ER vesicles from BY-2 cells. In the S12 fraction, small GFP-ER vesicles were dispersed (S12 fraction). After discontinuous sucrose density gradient centrifugation of S12 fraction, the interface between 1.0M and 1.5 M sucrose was recovered and used as GFP-ER vesicles in this study (GFP-ER vesicle). Bars = 10 μm.

3 Fig. S3 Elongation of tubules from GFP-ER vesicles. Numbers on upper right are the times in seconds from recording the images. The arrows indicate the tips of elongating tubules. The velocity of tubular elongation in A, B, C, and D was estimated to be about 3.2, 11, 50 and 40 μm/sec, respectively. The elongated tubule in E retracted with a velocity at 11.3 μm/sec from 0 to 0.4 sec. Bar = 10 μm.

4 Fig. S4 The presence of actin binding protein, villin, in the S12 fraction, and the arrangement of added F-actin into bundles. A, Immunoblotting of the S12 fraction with villin specific antibodies. The molecular masses of standard proteins are indicated on the left in kilodaltons. Ba, S12 fraction stained with RP. Only a few actin filaments and very short actin bundles were found. b, RP-F-actin in a homogenization solution. c, Bundles of RP-F-actin in the S12 fraction. Bar = 10 μm.

5 Fig. S5 Reduction of endogenous actin concentrations in S12 fraction by DNase I beads. A, Immunoblotting of chicken actin at concentrations of 5.6 (a), 11.2 (b), 28 (c), 56 nm (d), 4-fold diluted S12 fraction (e), 4-fold diluted S12 fraction after treatment with BSA-beads (f), and the S12 fraction after treatment with DNase I beads (g), with the antibody against actin. B, The relation between actin concentration and band intensity measured from Aa to Ad. The arrows e, f and g indicate band intensities of Ae, Af and Ag, respectively. In this example from five separate experiments, the concentration of four-fold diluted S12 fraction without or with the treatment of BSA beads was estimated to be 54 or 52.3 nm, respectively, while those of S12 fraction treated with DNase I beads to be 16 nm. C, ER tubule formation in the S12 fraction after the treatment with the BSA beads (C) or the DNase I beads (D) in the presence of 2.5 g/ml RP-F-actin, 2 mm ATP and 0.5 mm GTP. Bar = 10 μm.

6 Fig. S6 Exogenous microtubules did not induce the formation of ER tubules in the S12 fraction. A, Microtubules stained with Oregon-Green 488 taxol. B, The S12 fraction with taxol stabilized microtubules, ATP and GTP. C, S12 fraction with F-actin, ATP and GTP. Extensive formation of ER tubules and their networks were induced in the presence of F-actin (C), whereas few ER tubules were found in the presence of microtubules (B). Bars = 10 μm.

7 Fig. S7 Two classes of myosin in the S12 fraction of BY-2 cells. A, Immunoblotting of Arabidopsis seedlings with anti-atm1 antibody (b). a, CBB staining pattern. B, Immunoblotting of BY-2 S12 fraction with anti-atm1 (b), anti-bm175 tail (c) and anti-bm175 peptide antibody (d). a, CBB staining pattern. C, D and E, The CBB staining gel (a) and the immunoblot (b) of the immunoprecipitates with the anti-atm1, anti-bm175 tail and anti-bm175 peptide antibody, respectively. The immunoprecipitates in the S12 fraction with each myosin antibody was applied on SDS-PAGE (a) and then immunoblotted with each myosin antibody (b). The crossreacting bands, 130-kD and175-kd polypeptides indicated by arrows in C and D or E were excised and subjected to MS analysis. The molecular masses of standard proteins are indicated on the left in kilodaltons.

8 Fig. S8 MS analysis of immunoprecipitates with myosin antibodies in the S12 fraction. The sequences of peptides from 130-kD polypeptide as shown in panel a in Supplemental Figure S7C closely matched those in the Nb myosin VIII-1 heavy chain (A; red fonts), while those from 175-kD polypeptides as shown in panel a in Supplemental Figure S7D or S7E closely matched those in the Nb myosin XI-2 (B or C) and the 175-kD myosin heavy chains (B or C).

9 Fig. S9 Dissociation of BY-2 myosin VIII-1 from F-actin by ATP. The co-precipitant with F-actin (a) in the crude extract prepared from BY-2 cells was treated with a low ionic strength solution (-KCl) containing ATP. After centrifugation, the supernatant (b) and the pellet including F-actin (c) was recovered. Then the pellet was treated with a high ionic strength solution (+0.3 M KCl) without ATP. After centrifugation, the supernatant (d) and the pellet (e) were recovered, and the pellet was further treated with ATP-containing high ionic strength solution. After centrifugation, the supernatant (f) and the pellet (g) were separated. A and B, Immunoblotting with the antibody against 175-kD myosin heavy chain and the anti-atm1 antibody, respectively. The 175-kD myosin bound to F-actin was dissociated from F-actin by the ATP-containing low ionic strength solution (lane b in A), whereas the BY-2 myosin VIII-1 was released from F-actin by the ATP-containing high ionic strength solution (lane f in B). The molecular masses of standard proteins are indicated on the left in kilodaltons.

10 Fig. S10 Preparation of BY-2 myosin VIII-1 from the eluate of ATP-containing high ionic strength solution by a hydroxylapatite (A) and then DEAE Sephacel (B) column chromatography. A, Immunoblotting of fractions eluted from hydroxylapatite column over a linear concentration gradient of 100 mm to 350 mm potassium phosphate buffer, ph 7.0, (dashed line), with the anti-atm1 antibody (upper panel) or the antibody against 175-kD myosin heavy chain (lower panel). The BY-2 myosin VIII-1 was eluted at above 250 mm potassium phosphate buffer, while the 175-kD myosin at 120 to 200 mm. The fractions containing BY-2 myosin VIII-1 (Fraction 20 to 26) were further applied to the DEAE Sephacel column and eluted with a linear gradient of 0 to 600 mm KCl. B, Immunoblotting of fractions in DEAE Sephacel column with the anti-atm1 antibody. Dashed line indicates the concentration of KCl. The molecular masses of standard proteins are indicated on the left in kilodaltons.

11 Fig. S11 DEAE Sephacel fraction containing the BY-2 myosin VIII-1 and immuno-precipitation with the anti-atm1 antibody. Aa, Silver staining pattern of fraction 24 in DEAE Sephacel column shown in Supplemental Figure S7B. b and c, Immunoblotting this fraction with anti-atm1 antibodies and the antibody against 175-kD myosin heavy chain, respectively. B, Immuno-depletion of fraction with the antibody against 175-kD myosin (a and b) or the anti-atm1 antibody (c and d). Fraction 24 was mixed with protein-a beads pretreated with each antibody. After centrifugation, the supernatant (a or c) and the pellet (b or d) containing beads were recovered. The supernatant was used for an in vitro motility assay as shown in Supplemental Figure S12. About 80% of BY-2 myosin VIII-1 containing in fraction 24 was immuno-precipitated with the anti-atm1 antibody (d), whereas not with the antibody against the 175-kD myosin heavy chain (b). The molecular masses of standard proteins are indicated on the left in kilodaltons.

12 Fig. S12 Motile activity in vitro in the supernatant after immuno-precipitation. A, The motile activity in the supernatant shown in lane a in Supplemental Figure S11B. a, Initial image (0 sec). b, After 2 sec. c, Merged image of a and b. Arrows indicate the non-motile RP-F-actins showing saltatory or Brownian motion, while other RP-F-actins show the sliding movement. B, The motile activity in the supernatant shown in lane c in Supplemental Figure S11B. a, Initial image (0 sec). b, After 8 sec. c, Merged image of a and b. In this fraction, in which about 80% of BY-2 myosin VIII-1 was removed, most RP-F-actins show the saltatory or Brownian motions. Bar = 10 μm.

13 Fig. S13 The immno-depletion of 175-kD myosin with the anti-bm175 peptide antibody from the S12 fraction. The S12 fraction (S12) was mixed with the anti-atm1 (b) or the anti-bm175 peptide antibody (c) and then with protein-a beads. After centrifugation, the supernatant (Sup) and pellet (Ppt) were recovered and then subject to immunoblotting with the anti-bm175 tail antibody. As a control, the S12 fraction was mixed only with protein-a beads and then immunoblotting was carried out (a). B and C, The formation of ER tubules in the Sup shown in Ab and Ac, respectively, in the presence of 2 mm ATP, 0.5 mm GTP and 2.5 μg/ml RP-F-actin. Bar = 10 μm.

14 Fig. S14 A recombinant C-terminal tail region of 175-kD myosin heavy chain. A, Purity of recombinant proteins of C-terminal 100 amino acids (recombinant tail protein) of 175-kD myosin (a) and control polypeptide (control recombinant protein; b) fused to the dihydrofolate reductase. The molecular masses of standard proteins are indicated on the left in kilodaltons. B, Effect of recombinant tail protein on the association of 175-kD myosin with microsomes. The S12 fraction (S12) was mixed without (a) or with 20 μg/ml (b) recombinant tail protein. After ultra-centrifugation, the supernatant (Sup) and pellet (Ppt) were recovered and then immunoblotting was carried out with the antiserum against 175-kD myosin heavy chain. The amount of 175-kD myosin in microsome fraction (Ppt in b) was reduced to below 9% by the addition of recombinant tail protein. C, Effect of control recombinant protein on the association of 175-kD myosin with microsomes. The S12 fraction with the control recombinant protein at concentration of 25 μg/ml was treated in the same manner as described above. The amount of 175-kD myosin in the pellet was not altered with (Ppt in b) or without the control recombinant protein (Ppt in a). D, Effect of recombinant tail protein on the association BY-2 myosin VIII-1. The S12 fraction with the recombinant tail protein at concentration of 20 μg/ml was treated in the same manner as described above, instead of immunoblotting with the anti-atm1 antibody. The amount

15 of BY-2 myosin VIII-1 in the pellet was not altered with (Ppt in b) or without (Ppt in a) the recombinant tail protein.