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First published online 29 January 2008
doi: 10.1242/jcs.019281

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LUMA interacts with emerin and influences its distribution at the inner nuclear membrane

Luiza Bengtsson^* and Henning Otto

Freie Universität Berlin, Institut für Chemie und Biochemie, Thielallee 63, 14195 Berlin, Germany

View larger version (29K): [in this window] [in a new window]   Fig. 1. LUMA orthologs and expression of LUMA mRNA in human tissues. (A) Some BLASTP search hits with murine LUMA as a template. (B) Organization of predicted domains in selected orthologous LUMA sequences. Black boxes represent TMDs. (C) Multiple-tissue panels of first-strand cDNAs were tested for LUMA and, as a control, for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression by semi-quantitative reverse transcriptase (RT)-PCR.

View larger version (45K): [in this window] [in a new window]   Fig. 2. LUMA is an ER protein enriched at the INM. (A) Localization of LUMA in relation to emerin or lamin A/C in HeLa and NIH 3T3 cells, visualized by immunofluorescence staining. LUMA colocalizes with emerin at the NE and frames lamin A/C. A non-nuclear ER pool of LUMA is indicated by arrows. Overlay images show LUMA in green, emerin or lamin A/C in red. Scale bars, 5 µm. (B) Wild-type (MEF LMNA+/+) and LMNA knockout (MEF LMNA–/–) MEFs were stained with anti-LUMA antibody. Scale bars, 5 µm. (C) Immunoblots of proteins extracted from MEF LMNA–/– and LMNA+/+ cells were stained for LUMA, lamin A/C and, as a loading control, tubulin. (D) Untreated HeLa cells or HeLa cells transiently expressing LUMA 1-400 were extracted with Triton X-100 prior to fixation. Emerin and either endogenous LUMA (HeLa) or LUMA 1-400 carrying a V5-tag (HeLa/LUMA 1-400) were visualized by immunofluorescence staining. (E) Scheme of subcellular fractionation employed for detecting LUMA enrichment at the NE. (F) Whole-cell homogenate (H), nuclei (N), nuclear envelopes (NE) and ER membranes (ER) were prepared from N2a cells and immunoblotted for LUMA, emerin, LAP2 membrane isoforms, lamin A/C, calnexin and Sec61 (G) Whole cells, isolated nuclei and nuclear envelopes were extracted with Triton X-100 under low-salt (0.1 M NaCl) or high-salt (2 M NaCl) conditions. Solubilized (S) and pelleted (P) proteins were immunoblotted for LUMA.

View larger version (29K): [in this window] [in a new window]   Fig. 3. LUMA structure prediction. (A) Kyte-Doolittle hydropathy plot. The question mark indicates part of the sequence that is predicted to form a TMD although the hydropathy profile does not clearly show a hydrophobic-sequence stretch. (B) Scheme of predicted domains in LUMA. Numbers indicate first and last amino acid of each predicted feature. Black and hatched boxes indicate putative TMDs. The grey box marks the PFAM domain DUF 1625. (C) Averaged secondary-structure prediction for LUMA according to analysis with the programs GOR1, NNPREDICT, HNN, SOPM and SOPMA. Grey, -helices; black, β-sheets (see also supplementary material Fig. S3). (D) Prediction of natively unfolded amino-acid stretches for LUMA. Grey, folded regions; black, natively unfolded regions. Numbers above indicate beginning and end of the large predicted natively unfolded region.

View larger version (23K): [in this window] [in a new window]   Fig. 4. LUMA topology. (A) Model for LUMA topology with cytoplasmic or nucleoplasmic termini and a large lumenal hydrophilic domain. (B) Scheme of LUMA constructs: full-length LUMA 1-400 bears a C-terminal V5-His6-tag, the other constructs contain a Myc-His6-tag. N-terminal truncation mutants starting with LUMA residue 53 received a N-terminal signal peptide (SP; grey box) for ER export. Anti-LUMA antibodies recognize the hydrophilic domain of LUMA (indicated by the bar above the scheme for LUMA 1-400), which is replaced by a part of β-galactosidase in the LUMAgal mutant (gal; grey box). Black boxes indicate TMDs. (C) Full-length LUMA and LUMA 1-309 were 35S-labeled by coupled in vitro transcription and translation in the presence of microsomes, and then digested with proteinase K. Remaining fragments were separated by SDS-PAGE and visualized by autoradiography. a, full-length LUMA 1-400 V5/His; b, 42.5-kDa fragment of LUMA protected from proteolysis by the membrane; c, LUMA 1-309 and fragment; d, by-product of LUMA 1-309 translation; e and f, fragments generated from LUMA not inserted into microsomal membranes.

View larger version (56K): [in this window] [in a new window]   Fig. 5. LUMA topology explored by immunofluorescence. (A) HeLa cells were permeabilized with either digitonin or Triton X-100 and stained for LUMA. Tubulin or lamin B2 were visualized as controls. (B) Comparison of antibody-epitope accessibility of digitonin- and Triton-X-100-permeabilized cells overexpressing full-length LUMA or the LUMAgal mutant. LUMA (hydrophilic domain) and either the V5- or Myc-tag were visualized by immunofluorescence staining as indicated. Mock, vector control. (B) Comparison of antibody-epitope accessibility of digitonin- and Triton-X-100-permeabilized cells overexpressing LUMA truncation mutants LUMA SP 53-345, LUMA SP 53-309, and LUMA SP 53-200. Emerin and the Myc-tag were visualized by immunofluorescence staining. (B,C) For structural features of the constructs refer to Fig. 4B. Scale bars, 5 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 6. LUMA oligomerization. (A) HeLa cells were transfected with full-length LUMA 1-400 together with truncation mutants. Full-length LUMA was immunoprecipitated with anti-V5 antibody, and the precipitate analyzed for the presence of truncation mutants with either anti-LUMA antibody (lanes 1-8) or anti-Myc antibody (lanes 9 and 10). LUMAe, endogenous LUMA; , higher molecular mass bands of LUMA; HC, IgG heavy chain; LC, IgG light chain; , LUMA 53-309 and a fragment from LUMA 1-309. (B) Full-length LUMA 1-400, LUMAgal and LUMA 53-309 were 35S-labeled by coupled in vitro transcription and translation in the presence of microsomes. The resulting hetero- and homooligomers were separated by BN gel electrophoresis in the presence or absence of SDS. The complexes were further resolved by second dimension SDS-PAGE and visualized by autoradiography. , SDS-resistant, low-molecular-mass complex of LUMA 1-400; , LUMA 1-400; , LUMAgal; , LUMA 53-309.

View larger version (44K): [in this window] [in a new window]   Fig. 7. Overexpression of LUMA has dominant-negative effects on NE structure. (A,B) V5-tagged full-length LUMA 1-400 (A) and Myc-tagged LUMA-truncation mutants (B) were overexpressed in HeLa cells and visualized by immunofluorescence with antibodies against V5 or Myc. The NE was stained by anti-emerin antibody, chromatin by DAPI. Panels labeled V5xDAPI and mycxDAPI are merged images of the respective individual images. Panels labeled Overlay show overlays of individual V5 or myc images with the emerin image (V5 or Myc, green; emerin, red; DAPI, blue). Scale bars, 5 µm. Additionally, a schematic representation of the LUMA variants is shown.

View larger version (50K): [in this window] [in a new window]   Fig. 8. Downregulation of LUMA. HeLa cells were transfected with either a control vector expressing a control miRNA or the vector construct Hmi 48 expressing a miRNA designed to knockdown human LUMA mRNA. 72 hours after transfection, cells were fixed and subjected to immunofluorescence staining of LUMA, emerin and lamin A/C. Transfected cells are easily detected by GFP coexpression. Scale bars, 5 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 9. LUMA interacts with emerin. (A) Immunoprecipitation of different endogenous proteins from undifferentiated C2C12 cells with specific antibodies as indicated. Stars indicate antibody cross-reactive proteins, arrowhead points at IgGs covering presumable LUMA signal. Input of emerin results from a longer exposure. (B) Full-length LUMA 1-400, LUMA 53-309 or LUMAgal were overexpressed in HeLa cells and immunoprecipitated with antibodies against their C-terminal tags (V5 or Myc). Precipitates were analyzed for the presence of LUMA and emerin. LUMAe, endogenous LUMA; , LUMA degradation product (lanes 1 and 3, masked in lane 2 by LUMA 53-309); , IgG heavy chains; , LUMA 53-309 in lane 5 and C-terminal fragment of LUMAgal in lane 6. (C) Model for LUMA-emerin complexes at the INM.