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. 2002 Mar;13(3):817-29.
doi: 10.1091/mbc.01-08-0402.

Identification of a novel light intermediate chain (D2LIC) for mammalian cytoplasmic dynein 2

Paula M Grissom  1 , Eugeni A Vaisberg, J Richard McIntosh
Affiliations

Identification of a novel light intermediate chain (D2LIC) for mammalian cytoplasmic dynein 2

Paula M Grissom et al. Mol Biol Cell. 2002 Mar.

Abstract

The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo.

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Figures

Figure 1
Figure 1
Identification and molecular analysis of human D2LIC. (A) Silver-stained gel of sucrose gradient fractions and immunoprecipitated complexes. COS-7 cell homogenate was fractionated on sucrose gradients and 15 S peak fractions pooled (lane 1). Immunoprecipitations from the 15 S fractions were performed using preimmune serum (lane 2) and DHC2 affinity-purified antibodies (lane 3). An ∼33-kDa polypeptide (arrow) that coimmunoprecipitated with DHC2 was excised from the gel and microsequenced. (B) DNA and predicted protein sequence for the human D2LIC. Two peptide fragments generated from microsequencing (underlined) were used to identify a clone (D2LIC) from the human dbEST database. The P-loop consensus motif is underlined and in bold. (C) Northern blot analysis. COS-7 poly(A+) RNA (lane 1), total COS-7 RNA (lane 2), and total HeLa RNA (lane 3) were size fractionated on a gel, transferred to nylon, and probed with the full-length D2LIC clone (shown in B). A predominant 1.6-kb message is evident.
Figure 2
Figure 2
Dynein 2 light intermediate chain is a member of a family. (A) ClustalW Multiple Sequence Alignment of D2LIC and cytoplasmic dynein 1 LICs: rat LIC1, chicken DLC-A (D1LIC1), and rat LIC2. (B) ClustalW Multiple Sequence Alignment of dynein 2 light intermediate chains from mouse, C. elegans, D. melanogaster, and the yeast GTPase Spg1p. BOXSHADE was used to illustrate amino acid identity (darkly shaded residues) and similarity (lightly shaded residues). Accession numbers are rat D1LIC1, AF181992 (Purohit et al., 1999; Tynan et al., 2000a); chicken DLC-A (D1LIC1), Q90828 (Gill et al., 1994); rat D1LIC2, Q62698 (Hughes et al., 1995); mouse locus AK008822, BAB25915 (M. musculus Genome Sequencing Consortium); C. elegans hypothetical protein F02D8.3, T20505 (C. elegans Genome Sequencing Consortium); D. melanogaster CG3769 gene product, AAF52775.1 (Adams et al., 2000); and Spg1p, T45541 (Schmidt et al., 1997).
Figure 3
Figure 3
Immunoprecipitation and sedimentation analysis of the heavy chain and light intermediate chain of dynein 2. (A) D2LIC antibody specificity. Affinity-purified antibodies to D2LIC were prepared and analyzed on Western blots. COS-7 cell homogenate was fractionated by SDS-PAGE (Coomassie-stained gel; lane 1), transferred to nitrocellulose, and probed with affinity-purified antibodies to D2LIC (lane 2). (B) Immunoprecipitation analysis. Immunoprecipitates were prepared from COS-7 cell lysate (lane 1) by using rabbit preimmune serum for DHC2 (lane 2), DHC2 affinity-purified antibodies (lane 3), rat preimmune serum for D2LIC (lane 4), and D2LIC affinity-purified antibodies (lane 5), and then analyzed by Western blotting. The top panel was probed with antibodies to DHC2, and the bottom panel was probed with antibodies to D2LIC. (C) Specificity of immunoprecipitated complexes. Western blots of D2LIC immunoprecipitates (Figure 3B, lane 5) and COS-7 lysate (20 μg/lane) (Figure 3B, lane 1) were probed with affinity-purified antibodies to DHC2 (left) and to DHC1 (right). (D) Sedimentation analysis. Fractionation of COS-7 cell homogenate on a sucrose gradient was performed as described in MATERIALS AND METHODS. COS-7 cell lysate and gradient fractions were analyzed by Western blot with affinity-purified antibodies to DHC2 (top) and D2LIC (bottom).
Figure 4
Figure 4
Tissue-specific distribution of D2LIC and DHC2. An immunoblot of multiple tissue samples from mouse was prepared and cut at the molecular weight markers indicated to the right. The panels were probed with antibodies to either DHC2, the 74.1k IC of dynein 1 (D1 IC74), tubulin, or D2LIC.
Figure 5
Figure 5
Localization of D2LIC and DHC2 to the Golgi apparatus in COS-7 Cells. COS-7 cells were costained with D2LIC affinity-purified antibodies (shown in red) (A and D) and DHC2 affinity-purified antibodies (shown in green) (B and E). The merged images are shown in C and F. The DNA in all cells is visualized with DAPI (shown in blue). Cells were also double labeled with D2LIC antibodies (shown in red) (G and J) and antibodies to the Golgi maker p58 (shown in green) (H and K). The corresponding merged images are shown in I and L. Bars, 10 μm.
Figure 6
Figure 6
Effect of nocodazole treatment on the localization of D2LIC and DHC2 to the Golgi. COS-7 cells were treated with 0.3% DMSO (A, C, E, and G) or 33 μM nocodazole dissolved in DMSO (B, D, F, and H) for 1 h at 37°C. Cells were then fixed as described in MATERIALS AND METHODS and double labeled with anti-D2LIC (A and B) and anti-DHC2 (C and D). Treated cells were also double labeled with anti-D2LIC (G and H) and with antibodies to the Golgi marker p58 (E and F). Bars, 10 μm.
Figure 7
Figure 7
Effect of brefeldin A on the localization of D2LIC and DHC2 to the Golgi. COS-7 cells were treated at 37°C with 0.4% ethanol (A, D, and G), 10 μg/ml BFA dissolved in ethanol (B, E, H, and J–L) for 30 min, or 10 μg/ml BFA for 30 min followed by recovery in the absence of BFA for 90 min (C, F, and I). After these treatments, cells were fixed and double labeled with anti-D2LIC (A–C) and anti-DHC2 (D–F) or stained with anti-p58 (G–I). DNA in all cells is visualized with DAPI (shown in blue). BFA-treated cells were also double labeled with anti-D2LIC (J) and anti-γ-tubulin (K). The corresponding merged image is shown in L. Bars, 10 μm.
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