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Identification of septin-interacting proteins and characterization of the Smt3/SUMO-conjugation system in Drosophila

Hsin-Pei Shih1,*, Karen G. Hales1,§, John R. Pringle1,2,3 and Mark Peifer1,3

1 Department of Biology, University of North Carolina, Chapel Hill, NC 27599 USA
2 Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599 USA
3 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 USA
* Present address: Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403 USA
§ Present address: Department of Biology, Davidson College, Davidson, NC 28035, USA



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  		Fig. 1. Immunoblot analyses using anti-DmUba2 and anti-DmSmt3 antibodies. (A) Extracts of wild-type embryos (see Materials and Methods) were immunoblotted using affinity-purified anti-DmUba2 antibodies (lane 1) or the mock-affinity-purified fraction from pre-immune serum (lane 2). (B) Yeast strain JD90-1A carrying plasmids pIS2-ts10 and YCpIF2-DmUba2 (see Materials and Methods) was grown in selective medium containing either 2% galactose (lane 1) or 2% glucose (lane 2) as the carbon source. Extracts were immunoblotted using affinity-purified anti-DmUba2 antibodies. (C) Extracts of wild-type embryos were prepared either with 20 mM NEM (lane 1) or without NEM (lane 2) (see Materials and Methods) and immunoblotted using affinity-purified anti-DmSmt3 antibodies. Molecular weight markers are indicated. The arrow indicates the free form of DmSmt3.

 


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  		Fig. 2. Conjugation of DmSmt3 to DmUba2 and DmUbc9 in vitro. Fly embryo extracts supplemented with ATP and Mg2+ were incubated with His6DmSmt3, His6DmUb, or no tagged protein, and proteins were isolated using Ni-NTA beads and analyzed by SDS-PAGE and immunoblotting (see Materials and Methods). (A,B) Immunoblotting using DmUba2-specific antibodies. SDS-PAGE was conducted under reducing (A) or nonreducing (B) conditions. In (A), a sample of unpurified lysate was also analyzed. The species migrating at ~55 kDa is presumably a breakdown product of DmUba2 (see also Fig. 1A,B). (C,D) Immunoblotting using DmUbc9-specific antibodies. SDS-PAGE was conducted under reducing (C) or nonreducing (D) conditions. In C, a sample of unpurified lysate was also analyzed. In addition to DmUbc9 (predicted molecular weight, ~18 kDa) (Joanisse et al., 1998Go), anti-DmUbc9 antibodies also recognize a polypeptide of ~25 kDa; as this species is evident both in the lysate and among proteins isolated with His6DmUb, it may be a DmUb-conjugating enzyme that is related to DmUbc9. Molecular weight markers are shown for each panel.

 


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  		Fig. 3. Coimmunoprecipitation of DmUba2 and DmUbc9. Immunoprecipitates were prepared from embryo lysates using the antibodies indicated (see Materials and Methods) and analyzed by immunoblotting using (A) DmUba2-specific antibodies or (B) DmUbc9-specific antibodies. The immunoprecipitation using anti-myc antibodies serves as a negative control. The dark band at >=20 kDa in (B) is presumably an allotypic form of rabbit IgG light chain in the anti-DmUbc9 antibodies that is recognized by the HRP-conjugated goat anti-rabbit-IgG.

 


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  		Fig. 4. Localization of DmUba2 during embryogenesis. (A-I) Localization of DmUba2 in syncytial-blastoderm embryos during nuclear cycle 10 (A-C), 11 (D-F), and 13 (G-I). Surface views of embryos in interphase (A1-A3, D1-D3, G1, and G2) or mitosis (A4, D4, and G3) are shown; optical sections parallel to the surface and through the middle of nuclei in interphase (B1-B3, E1-E3, H1, and H2) or mitosis (B4, E4, and H3) and medial longitudinal optical sections of embryos in interphase (C1-C3, F1-F3, I1, and I2) or mitosis (C4, F4, and I3) are also shown. Each vertical set of three images (e.g., A1, B1, and C1) shows the same embryo. Each row of interphase images (e.g., A1, A2, and A3) is ordered according to the presumed sequence of the embryos within interphase, with the earliest point on the left (e.g., A1), based on the assumption that DmUba2 must gradually reaccumulate in the nucleus after its dispersion during mitosis. (J,K) Localization of DmUba2 in older embryos in stage 9 (J) or 12 (K). Medial longitudinal optical sections are shown. Arrow indicates a mitotic domain. Bar, 10 µm (A-I); 50 µm (J and K).

 


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  		Fig. 5. Localization of DmUba2 and DmSmt3 during cellularization, in mitotic domains, and in cultured cells. Some cells are numbered for reference in the text. (A-D) Embryos early (A,B) or late (C,D) in cellularization were stained for Pnut (red) and either DmUba2 (green in A,C) or DmSmt3 (green in B,D). (E-G, I-M) Mitotic domains in extended-germband embryos were double stained for DmUba2 (E, E inset, and green in G and G inset) or DmSmt3 (K and green in I,J,M) and either Pnut (F,L, and red in G,I,M) or tubulin (F inset and red in G inset and J). Arrowheads in I,J indicate concentrations of Smt3 in the regions of the chromosomes. Images of three different embryos are shown in K-M. (H) Clone-8 cells undergoing cytokinesis were stained for DmSmt3 (green) and Pnut (red). Bars, 10 µm (A-G, same magnification; K-M same magnification).

 


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  		Fig. 6. Localization of DmUba2 and DmSmt3 at other developmental stages and in other cell types. (A-C) Localization of DmUba2 to nuclei during oogenesis. Egg chambers were double labeled with anti-DmUba2 and anti-Pnut. Arrows and arrowheads indicate structures discussed in the text. (A) Ovariole with the germarium on the left and successively older egg chambers moving left to right. DmUba2 (top panel; green in bottom panel) and Pnut (middle panel; red in bottom panel) are shown. (B) Overexposure of DmUba2 staining of an ovariole similar to that shown in A. (C) Higher magnification of a germarium, showing enrichment of DmUba2 (green) in nuclei of both follicle and germ cells, as well as in the nuclei of the muscle cells in the sheath surrounding the germarium. (D,E) Embryonic central nervous system after double staining for Pnut (red) and either DmUba2 (green in D) or DmSmt3 (green in E). (F,G) DmSmt3 localization to nuclei of syncytial blastoderm embryos during interphase (G) and to the chromosomes during mitosis (F). (H) Localization of DmSmt3 (green) to interphase nuclei during embryogenesis. Embryos at stages 8 (H1), 10 (H2), and 14 (H3) are shown.

 

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