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, 12 (7), 2195-206

Null Mutants of the Neurospora Actin-Related Protein 1 Pointed-End Complex Show Distinct Phenotypes

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Null Mutants of the Neurospora Actin-Related Protein 1 Pointed-End Complex Show Distinct Phenotypes

I H Lee et al. Mol Biol Cell.

Abstract

Dynactin is a multisubunit complex that regulates the activities of cytoplasmic dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end--binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Delta ro-2, Delta ro-7, and Delta ro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level dynein ATPase activity, and we find that all three mutants have low dynein ATPase activity. Surprisingly, Delta ro-12 differs from Delta ro-2 and Delta ro-7 and other previously characterized dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct dynein/dynactin localization pattern. All three mutants also show stronger dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.

Figures

Figure 1
Figure 1
Predicted amino acid sequences of N. crassa RO2 and RO12. (A) Alignment of the predicted amino acid sequence of N. crassa RO2 with p62 from rat and worm (C. elegans). (B) Alignment of the predicted amino acid sequence of N. crassa RO12 with p25 from mouse. Hyphens that interrupt sequences indicate gaps that were introduced to maximize the alignment. Dark and light shaded boxes indicate identical and similar amino acids, respectively.
Figure 2
Figure 2
Association of N. crassa RO2 with RO3 (p150Glued). (A) RO2 and RO3 were immunoprecipitated in separate experiments with the use of anti-RO2 and anti-RO3 antibodies, respectively. Immunoprecipitants were resolved by SDS-PAGE, and RO2 and RO3 proteins were detected by Western blotting. The cell extracts and antibodies (antibody) used in these experiments are indicated above the immunoblot. (B) Cell extracts from wild type (WT) and Δro-2 were fractionated by centrifugation on 10-ml 5–20% linear sucrose gradients. Fractions (500 μl) were collected from the bottom of each tube (labeled lanes 1 through 8), and proteins were concentrated by trichloroacetic acid fractionation and then subjected to SDS-PAGE. RO1 (dynein heavy chain), RO2, and RO3 were detected by Western blotting. Sedimentation standards were thyroglobulin (19S), catalase (11S), and alcohol dehydrogenase (5S).
Figure 3
Figure 3
The effect of Arp1 pointed-end null mutations (Δro-2, Δro-7, and Δro-12) on nuclear distribution relative to wild type (WT) and the dynein heavy chain [ro-1(B15)] and dynactin p150Gluedro-3) null mutants. DAPI staining for nuclear localization was performed as described in MATERIALS AND METHODS. Bar, 5 μm.
Figure 4
Figure 4
Vacuole distribution in Arp1 pointed-end null mutants (Δro-2, Δro-7, and Δro-12) relative to wild type (WT) and dynactin p150Gluedro-3) null mutant. Staining for vacuoles was performed as described in MATERIALS AND METHODS. Bar, 5 μm.
Figure 5
Figure 5
Immunolocalization of RO1 and RO3 in Arp1 pointed-end null mutations (Δro-2, Δro-7, and Δro-12) and wild type (WT). For Δro-2 mutant, staining for RO1 and RO3 was from tip and distal regions, for Δro-7 mutant, RO1 and RO3 staining was from only the distal region, and for Δro-12, RO1 and RO3 staining was from only hyphal tips. The N. crassa strains were labeled in the right side and proteins localized were labeled at the top of the figure. Samples were prepared for immunolocalization as described in MATERIALS AND METHODS. Bar, 5 μm.
Figure 6
Figure 6
Dynein ATPase activity in wild type (WT) and Δro-3, Δro-2, Δro-7, and Δro-12 mutants. Cell extracts from each strain were fractionated with the use of a Sepharose CL-4B (bed volume, 100 ml) column, and fractions volume from 28th to 37th were assayed for ATPase activity (see MATERIALS AND METHODS). Data are representative of three independent experiments. The values are within the range of 5% for each assay from three independent preparations.
Figure 7
Figure 7
Dynein and dynactin membrane association in wild type (WT) and Arp1 pointed-end complex mutants. (A) The abundance of dynein (RO1) and dynactin (RO3) was determined by Western blotting of low-speed cell extracts from wild type, Δro-12, Δro-2, and Δro-7. (B) Dynein- and dynactin-membrane binding was determined by salt-dependent pelleting and membrane-flotation experiments (see MATERIALS AND METHODS). N. crassa strains used are labeled on the left, and the RO1 and RO3 proteins detected by immunoblotting are labeled on the right. Lanes 1 and 2 are supernatant and pellet, respectively, after high-speed (100,000 × g) centrifugation of low-speed cell extracts. Lanes 3 and 4 are supernatant and pellet, respectively, after 100,000 × g centrifugation of 100 mM KCl-treated low-speed cell extract. Lanes 5 and 6 are supernatant and pellet, respectively, after 100,000 × g centrifugation of 200 mM KCl-treated low-speed cell extract. Lanes 7 and 8 are supernatant and pellet, respectively, after 100,000 × g centrifugation of supernatant without the addition of pellet from lanes 5 and 6. Lanes 9 and 10 are supernatant and pellet, respectively, after 100,000 × g centrifugation of recombined desalted supernatant and pellet from lanes 5 and 6. Lanes 11 and 12 are 1.4 M sucrose and 1.4/0.25 M sucrose interface after flotation of membranes contained within the pellet fraction present in lane 10 (see MATERIALS AND METHODS). Rebinding experiments were conducted in either the absence (−M) or presence (+M) of membrane fraction.

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