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Additional file 4. Supplemental Methods.

Additional file 5. Uncropped versions of all blots.

Additional file 6. Review history.

Additional file 2: Figure S1. (Fig. 3 continued). LAP2�� promotes the loading of RPA onto damaged chromatin. Figure S2. (Fig. 5 continued). LAP2��-promoted RPA loading is required for ATR activation and homologous recombination. Figure S3. (Fig. 6 continued). LAP2 is engaged into damaged chromatin in a PARP1-dependent manner.

Background: Atypical degradative polyubiquitylation on non-lysine residues has only been reported in metazoans. Results: Lysine-less mutant of Asi2 inner nuclear membrane protein is ubiquitylated and targeted to proteasomes in a Doa10-, Ubc6-, and Ubc7-dependent manner. Conclusion: Well characterized enzymes of the endoplasmic reticulum-associated degradation pathway can catalyze atypical ubiquitylation. Significance: Atypical degradative ubiquitylation is not restricted to metazoans and represents an unexplored process in yeast., Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to ϵ-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.

Highlights • A-type lamins localize to the lamina at the nuclear periphery and throughout the nucleoplasm. • A-type lamins in the nuclear interior bind to and depend on lamina-associated polypeptide 2α (LAP2α). • Lamin A/C–LAP2α complexes regulate tissue progenitor cell proliferation through retinoblastoma protein-linked pathways. • A-type lamins regulate chromatin organization and gene expression at the nuclear periphery and throughout the nucleoplasm. • Deregulation of nucleoplasmic A-type lamins and LAP2α contributes to abnormal phenotypes in laminopathies., A-type lamins are components of the lamina network at the nuclear envelope, which mediates nuclear stiffness and anchors chromatin to the nuclear periphery. However, A-type lamins are also found in the nuclear interior. Here we review the roles of the chromatin-associated, nucleoplasmic LEM protein, lamina-associated polypeptide 2α (LAP2α) in the regulation of A-type lamins in the nuclear interior. The lamin A/C–LAP2α complex may be involved in the regulation of the retinoblastoma protein-mediated pathway and other signaling pathways balancing proliferation and differentiation, and in the stabilization of higher-order chromatin organization throughout the nucleus. Loss of LAP2α in mice leads to selective depletion of the nucleoplasmic A-type lamin pool, promotes the proliferative stem cell phenotype of tissue progenitor cells, and delays stem cell differentiation. These findings support the hypothesis that LAP2α and nucleoplasmic lamins are regulators of adult stem cell function and tissue homeostasis. Finally, we discuss potential implications of this concept for defining the molecular disease mechanisms of lamin-linked diseases such as muscular dystrophy and premature aging syndromes.

Ankle1 does not accumulate on condensed chromosomes during mitosis. U2OS cells stably expressing GFP-Ankle1mut were fixed, stained with DAPI and imaged using a LSM700 confocal microscope. Representative images of at least five cells per mitotic phase from three independent experiments are shown. Scale bar: 10Â Îźm. (PDF 1788 kb)

Catalytically dead Ankle1 mutant shuttles like wild-type Ankle1 but does not induce DNA damage response. (A) U2OS cells stably expressing GFP-Ankle1mut (GIY-YIG to GIY-AAA) were imaged using confocal microscopy without or after 3 h leptomycin B treatment. (B) U2OS cells were transiently transfected with Ankle1wt-V5 or Ankle1mut-V5 (GIY-YIG to GIY-AAA). Cells were analyzed by confocal immunofluorescence microscopy following staining with antibodies to V5 and γH2A.X. Scale bars: 10 μm. (PDF 990 kb)

The nuclear envelope is a barrier comprising outer and inner membranes that separate the cytoplasm from the nucleoplasm. The two membranes have different physical characteristics and protein compositions. The processes governing the stability of inner nuclear membrane (INM) proteins are not well characterized. In Saccharomyces cerevisiae, the INM Asi1-Asi3 complex, principally composed of integral membrane proteins Asi1 and Asi3, is an E3 ubiquitin ligase. In addition to its well-documented function in endoplasmic reticulum (ER)-associated degradation, the Doa10 E3 ubiquitin ligase complex partially localizes to the INM. The Asi1-Asi3 and Doa10 complexes define independent INM-associated degradation (INMAD) pathways that target discrete sets of nuclear substrates for proteasomal degradation. Here, we report that Asi1 is rapidly turned over (t1/2≤30 min). Its turnover depends on ubiquitin-mediated degradation by nucleus-localized proteasomes, exhibiting a clear requirement for the E2 ubiquitin-conjugating enzyme Ubc7, Cue1 and the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs largely independently of the Asi1-Asi3 or Doa10 complexes, indicating that it is subject to quality control at the INM in a manner distinct from that of the characterized INMAD pathways.

An intricate machinery protects cells from the accumulation of misfolded, non-functional proteins and protein aggregates. Protein quality control pathways have been best described in the cytoplasm and the endoplasmic reticulum, however, recent findings indicate that the nucleus is also an important compartment for protein quality control. Several nuclear ubiquitinylation pathways target soluble and membrane proteins in the nucleus and mediate their degradation through nuclear proteasomes. In addition, emerging data suggest that nuclear envelope components are also degraded by autophagy, although the mechanisms by which cytoplasmic autophagy machineries get access to nuclear targets remain unclear. In this minireview we summarize the nuclear ubiquitin-proteasome pathways in yeast, focusing on pathways involved in the protein degradation at the inner nuclear membrane. In addition, we discuss potential mechanisms how nuclear targets at the nuclear envelope may be delivered to the cytoplasmic autophagy pathways in yeast and mammals.

DNA damage response is active in U2OS cells stably overexpressing GFP-Ankle1mut. Cells were fixed and stained for γH2A.X either untreated or after overnight treatment with bleomycin (1 μg/mL), mitomycin C (0.5 μg/mL) or hydroxyurea (100 μM) and imaged using confocal microscopy. Scale bar: 10 μm. (PDF 976 kb)

Laminopathies are rare genetic disorders caused by mutations in genes encoding lamins or lamin-interacting proteins. LMNA, EDM and to a lower measure the Nesprin genes have been associated with Emery Dreifuss muscular dystrophy (EDMD), characterized by contractures of the Achilles tendons, progressive skeletal muscle weakness and heart rhythm disturbances leading to dilated cardiomyopathy (DCM) and sudden cardiac death. Since ~60% EDMD patients are not associated to known genes, we used a functional candidate-gene approach to identify additional EDMD disease genes. Based on reported interactions of lamina associated polypeptide 2 (LAP2) with nucleoplasmic lamin A/C and the association of the LAP2alpha isoform to DCM (Matthew R.G Taylor et al. 2005), 111 EDMD and 87 DCM patients were investigated for DNA variations in LAP (encoding six LAP2 isoforms) using heteroduplex analysis and direct sequencing. Among ten variations found, four changes—p.P426L (c.1481C>T) in LAP2alpha, p.D271E (c.1054T>G) in LAP2beta, and p.V423L (c.1058G>C) and p.M381I (c.1387 G>A) in LAP2gamma—were unique for EDMD patients, but segregation analysis indicated only p.P426L LAP2alpha as a mutation potentially associated to EDMD. P426L-LAP2 alpha localized to the nucleoplasm in skin fibroblasts, like wild-type protein and did not affect the loalization of lamin A. However, in mutant fibroblast cultures phosphorylated retinoblastoma protein (Rb) levels were reduced compared to wild-type cultures, sugesting that the mutated protein may affect cell cycle progression in agreement with previous studies implicating LAP2alpha-lamin A in Rb-mediated cell cycle control. The present study shows that LAP2alpha mutations might add to the pathology of EDMD in ~1% of EDMD patients.

The intermediate filament proteins, A- and B-type lamins, form the nuclear lamina scaffold adjacent to the inner nuclear membrane. B-type lamins confer elasticity, while A-type lamins lend viscosity and stiffness to nuclei. Lamins also contribute to chromatin regulation and various signaling pathways affecting gene expression. The mechanical roles of lamins and their functions in gene regulation are often viewed as independent activities, but recent findings suggest a highly cross-linked and interdependent regulation of these different functions, particularly in mechanosignaling. In this newly emerging concept, lamins act as a "mechanostat" that senses forces from outside and responds to tension by reinforcing the cytoskeleton and the extracellular matrix. A-type lamins, emerin, and the linker of the nucleoskeleton and cytoskeleton (LINC) complex directly transmit forces from the extracellular matrix into the nucleus. These mechanical forces lead to changes in the molecular structure, modification, and assembly state of A-type lamins. This in turn activates a tension-induced "inside-out signaling" through which the nucleus feeds back to the cytoskeleton and the extracellular matrix to balance outside and inside forces. These functions regulate differentiation and may be impaired in lamin-linked diseases, leading to cellular phenotypes, particularly in mechanical load-bearing tissues.

The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of ∼45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post- translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.