NVL2 is a nucleolar AAA-ATPase that interacts with ribosomal protein L5 through its nucleolar localization sequence - PubMed (original) (raw)

NVL2 is a nucleolar AAA-ATPase that interacts with ribosomal protein L5 through its nucleolar localization sequence

Masami Nagahama et al. Mol Biol Cell. 2004 Dec.

Abstract

NVL (nuclear VCP-like protein), a member of the AAA-ATPase family, is known to exist in two forms with N-terminal extensions of different lengths in mammalian cells. Here, we show that they are localized differently in the nucleus; NVL2, the major species, is mainly present in the nucleolus, whereas NVL1 is nucleoplasmic. Mutational analysis demonstrated the presence of two nuclear localization signals in NVL2, one of which is shared with NVL1. In addition, a nucleolar localization signal was found to exist in the N-terminal extra region of NVL2. The nucleolar localization signal is critical for interaction with ribosomal protein L5, which was identified as a specific interaction partner of NVL2 on yeast two-hybrid screening. The interaction of NVL2 with L5 is ATP-dependent and likely contributes to the nucleolar translocation of NVL2. The physiological implication of this interaction was suggested by the finding that a dominant negative NVL2 mutant inhibits ribosome biosynthesis, which is known to take place in the nucleolus.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Schematic representation of human NVL1 and NVL2. The numbering of amino acids starts from the initiation methionine of NVL2. The translation of NVL1 starts from the residue corresponding to the second methionine (residue 107) of NVL2. The amino acid sequences of potential nuclear and nucleolar localization signals are indicated. The residues mutated in this study are underlined, and named M1, M2, M3, and M4, respectively.

Figure 2.

Figure 2.

Differential subnuclear localization of NVL isoforms. (A) A 293T cell lysate was analyzed by Western blotting using an anti-NVL antibody. The molecular size markers are indicated on the left. (B) HeLa cells were untreated or treated with ActD (50 ng/ml) for 3 h. The localization of endogenous NVL proteins and nucleolin was determined by fluorescence microscopy after double staining with anti-NVL and anti-nucleolin antibodies. (C) Immunofluorescence analysis of the localization of NVL isoforms. HeLa cells were transiently transfected with FLAG-tagged NVL1 and NVL2. After 20 h, the cells were fixed and double-stained with anti-FLAG and anti-nucleolin antibodies. Nuclei were visualized with DAPI (DNA). Bars, 20 μm.

Figure 3.

Figure 3.

Subcellular localization of each domain of NVL2. HeLa cells were transiently transfected with full-length FLAG-tagged NVL2 or a truncation mutant representing the N- (residues 1–220), D1- (residues 221–553), D2- (residues 554–856), or ND1- (N- and D1-) domain. After 20 h, the cells were fixed and double-stained with anti-FLAG and anti-nucleolin antibodies. Bars, 20 μm.

Figure 4.

Figure 4.

Identification of a nuclear localization signal in NVL1. (A) HeLa cells were transiently transfected with the FLAG-tagged wild-type (WT) NVL1, NVL1-M3, NVL1-M4, and NVL1-M3/M4. After 20 h, the cells were fixed and double-stained with anti-FLAG and anti-nucleolin antibodies. Bars, 20 μm. (B) Quantitative analysis of the results in A. The cellular localization of the indicated constructs was scored as follows: cells showing more intense nuclear than cytoplasmic staining (N > C), cells showing a nearly equal distribution between the cytoplasm and nucleus (N = C), and cells showing predominantly cytoplasmic staining (N < C). At least 100 cells were scored for each construct.

Figure 5.

Figure 5.

Identification of nuclear and nucleolar localization signals in NVL2. (A) HeLa cells were transiently transfected with the FLAG-tagged wild-type (WT) NVL2, NVL2-M3/M4, NVL2-M1, NVL2-M2, and NVL2-M2/M3/M4. After 20 h, the cells were fixed and double-stained with anti-FLAG and anti-nucleolin antibodies. Bars, 20 μm. (B) Quantitative analysis of the results in A. At least 100 cells were scored for each construct.

Figure 6.

Figure 6.

L5 is an interaction partner of NVL2. (A) For yeast two-hybrid analysis, S. cerevisiae Y190 cells were transformed with bait vector pGBT9 containing NVL2, NVL1, or VCP/p97, or pGBT9 alone as a control (Empty), and prey vector pACT2 containing L5 (residues 81–297) or pACT2 alone as a control (Empty). The interaction was examined by monitoring β-galactosidase activity on a filter. Five independent transformants were examined for each pair of constructs. (B) A 293T cell lysate was analyzed by Western blotting using an anti-L5 antibody. The molecular size markers are indicated on the left. (C) The localization of endogenous L5 and nucleolin was determined by fluorescence microscopy after double-staining with anti-L5 and anti-nucleolin antibodies. Bars, 20 μm.

Figure 7.

Figure 7.

NVL2 interacts with L5 in an ATP-dependent manner. (A) 293T cells were transfected with FLAG-tagged forms of NVL1 or NVL2. After 24 h, cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody in the presence or absence (Control) of ATP. The immunoprecipitates (IP) and 2% of the starting material (Input) were resolved by SDS-PAGE and then analyzed by Western blotting. (B) A cell lysate of 293T cells expressing GST-L5 or GST as a control was subjected to GST pull-down assay with glutathione beads. ATP was included during the incubation. The proteins bound to glutathione beads (Pull-down) and 2% of the starting material (Input) were resolved by SDS-PAGE and then analyzed by Western blotting.

Figure 8.

Figure 8.

The NoLS of NVL2 is required for its interaction with L5. (A) For yeast two-hybrid analysis, S. cerevisiae Y190 cells were transformed with bait vector pGBT9 containing the indicated NVL2 domain, or pGBT9 alone as a control (Empty), and prey vector pACT2 containing L5 (residues 81–297) or pACT2 alone as a control (Empty). The interaction was examined by monitoring β-galactosidase activity on a filter. Five independent transformants were examined for each pair of constructs. full, full-length. (B) 293T cells were transfected with the indicated FLAG-tagged mutant of NVL1 or NVL2. After 24 h, cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody. ATP was included during the incubation. The immunoprecipitates (IP) and 2% of the starting material (Input) were resolved by SDS-PAGE and then analyzed by Western blotting.

Figure 9.

Figure 9.

N-terminally truncated L5(81–297) relocalizes NVL2 from the nucleolus to nucleoplasmic punctate structures. (A) HeLa cells were transiently transfected with L5-GFP or L5(81–297)-GFP. After 20 h, the cells were fixed and stained with an anti-NVL antibody (panels b and e) or anti-nucleolin antibody (panel h). L5-GFP (panel a) or L5(81–297)-GFP (panels d and g) was visualized directly. Merged images are shown in the right panels. (B) HeLa cells were transfected with FLAG-tagged forms of the wild-type (WT) NVL2 and the NVL2-M2/M3/M4 mutant. After 20 h, the cells were fixed and double-stained with an anti-FLAG antibody and anti-L5 antibody. Merged images are shown in the right panels. Bars, 20 μm.

Figure 10.

Figure 10.

Overexpression of the K628M mutant of NVL2 impairs 60S subunit biogenesis. (A) 293T cells were transfected with FLAG-tagged forms of the wild-type NVL2 (WT) and the K628M mutant. After 24 h, cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody in the presence of ATP. The immunoprecipitates (IP) and 2% of the starting material (Input) were resolved by SDS-PAGE and then analyzed by Western blotting. (B) The ribosome profiles (A254 nm) on sucrose gradient centrifugation of extracts of cells transfected with the control vector and the expression plasmids for the FLAG-tagged wild-type NVL2 (WT) and K628M mutant are shown. The positions of the 40S, 60S, and 80S ribosomal subunits are indicated.

Similar articles

Cited by

References

    1. Bernardi, R., Scaglioni, P.P., Bergmann, S., Horn, H.F., Vousden, K.H., and Pandolfi, P.P. (2004). PML regulates p53 stability by sequestering Mdm2 to the nucleolus. Nat. Cell Biol. 6, 665-672. - PubMed
    1. Carmo-Fonseca, M., Mendes-Soares, L., and Campos, I. (2000). To be or not to be in the nucleolus. Nat. Cell Biol. 2, E107-E112. - PubMed
    1. Claussen, M., Rudt, F., and Pieler, T. (1999). Functional modules in ribosomal protein L5 for ribonucleoprotein complex formation and nucleocytoplasmic transport. J. Biol. Chem. 274, 33951-33958. - PubMed
    1. Dechampesme, A.M., Koroleva, O., Leger-Silvestre, I., Gas, N., and Camier, S. (1999). Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway. J. Cell Biol. 145, 1369-1380. - PMC - PubMed
    1. Dingwall, C., and Laskey, R.A. (1991). Nuclear targeting sequences—a consensus? Trends Biochem. Sci. 16, 478-481. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources