Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

Batsios, Petros; Ren, Xiang; Baumann, Otto; Larochelle, Denis A.; Gräf, Ralph

doi:10.3390/cells5010013

Cited by 18 publications

(33 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Plasmids expressing the BirA*‐NE81 fusion were integrated in single or multicopy into the D. discoideum genome and high‐level fusion protein expression was induced by growth in axenic medium. Following purification of biotinylated proteins, immunoblotting revealed proximity labeling by BirA*‐NE81 of endogenous NE81, the lamin‐binding protein SUN1 , and the MAN1‐like protein Src1 . Additional proteins specifically biotinylated in the presence of BirA*‐NE81, but not in the control cells expressing BirA* alone, were also visualized in these experiments but as yet no analysis of these potentially novel interactors has been published.…”

Section: Bioid In Unicellular Organismsmentioning

confidence: 98%

Meet the neighbors: Mapping local protein interactomes by proximity‐dependent labeling with BioID

2016

View full text Add to dashboard Cite

Proximity‐dependent biotin identification (BioID) is a recently developed method that allows the identification of proteins in the close vicinity of a protein of interest in living cells. BioID relies on fusion of the protein of interest with a mutant form of the biotin ligase enzyme BirA (BirA*) that is capable of promiscuously biotinylating proximal proteins irrespective of whether these interact directly or indirectly with the fusion protein or are merely located in the same subcellular neighborhood. The covalent addition of biotin allows the labeled proteins to be purified from cell extracts on the basis of their affinity for streptavidin and identified by mass spectrometry. To date, BioID has been successfully applied to study a variety of proteins and processes in mammalian cells and unicellular eukaryotes and has been shown to be particularly suited to the study of insoluble or inaccessible cellular structures and for detecting weak or transient protein associations. Here, we provide an introduction to BioID, together with a detailed summary of where and how the method has been applied to date, and briefly discuss technical aspects involved in the planning and execution of a BioID study.

show abstract

Section: Bioid In Unicellular Organismsmentioning

confidence: 98%

Meet the neighbors: Mapping local protein interactomes by proximity‐dependent labeling with BioID

2016

View full text Add to dashboard Cite

show abstract

“…As in metazoan lamins, an α-helical, central rod domain consisting of 370 amino acid residues is preceded by a head domain including Dictyostelium nuclear envelope 511 a CDK1 phosphorylation consensus sequence and followed by (in this order) a tail domain featuring a NLS, a conserved lamin tail domain, and a CaaX-box (= cysteine, two aliphatic aa and X = residue specifying the type of isoprene moiety) for prenylation at the C-terminal end (Krüger et al, 2012). Furthermore, microscopic and molecular studies confirmed that NE81 should be considered as a bona fide lamin due to its localization, functions, and protein interactions Batsios et al, 2016a;Batsios et al, 2016b;Grafe et al, 2019;Krüger et al, 2012). For example, immunogold-electron microscopy revealed an exclusive distribution of NE81 along the inner nuclear envelope.…”

Section: The Amoebozoan Laminmentioning

confidence: 94%

“…The latter represents the only LEMfamily protein in Dictyostelium (see below and Fig. 1) (Batsios et al, 2016b). Interactions between these protein families are conserved at least in amoebozoans and opisthokonts suggesting that they are very ancient and were possibly already a feature of the LECA.…”

Section: The Amoebozoan Laminmentioning

confidence: 98%

“…In Dictyostelium the only HeH-family protein, the MAN1-like Src1, has been thoroughly characterized. Src1, as revealed by light and electron microscopy, is an integral INM protein that interacts with the lamin NE81 in BioID and mis-localization assays (Batsios et al, 2016b). Like several other HeH-family members in unicellular eukaryotes, Src1 contains no LEM-domain, probably due to the lack of its binding partner BAF (barrier to autointegration factor), which links the chromatin to the nuclear lamina (Shumaker et al, 2001).…”

Section: Heh-family Proteinsmentioning

confidence: 99%

“…Src1 may play a more significant role in the association of nucleolar patches with the nuclear envelope as it is concentrated there at the contact sites of nucleoli ( Fig. 3B) (Batsios et al, 2016b).…”

Section: Nucleolar Proteinsmentioning

confidence: 99%

See 2 more Smart Citations

Nuclear envelope organization in Dictyostelium discoideum

Batsios¹,

Gräf²,

Koonce³

et al. 2019

Int. J. Dev. Biol.

Self Cite

View full text Add to dashboard Cite

The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export. The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun1, as well as with the LEM/HeH-family protein Src1. Sun1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun1 usually forms a so-called LINC complex. Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in permeabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics. , and build upon the material for any purpose, even commercially), providing you give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. Printed in Spain Abbreviations used in this paper: BAF, barrier to autointegration factor; BioID, proximity-dependent biotin identification; CDK1, cyclin-dependent kinase 1; cryo-ET, cryo-electron tomography; DAPI, 4′,6-diamidin-2-phenylindol; EM, electron microscopy; ER, endoplasmic reticulum; ExM, expansion microscopy; feSEM, field-emission scanning electron microscopy; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; HeH, helix-extension-helix; IF, intermediate filament; INM, inner nuclear membrane; KASH, Klarsicht, ANC-1, Syne Homology; LBR, lamin B receptor; LECA, last eukaryotic common ancestor; LEM, lamina-associated polypeptide 2 (LAP2), Emerin, and MAN1; LINC, linker of the nucleoskeleton and the cytoskeleton; MCAK, mitotic centromere-associated kinesin; MEF, mouse embryonic fibroblast; NET, nuclear envelope transmembrane protein; NLS, nuclear localization sequence; NPC, nuclear pore complex; ONM, outer nuclear membrane; STED, stimulated emission depletion. import and export. On its nuclear side, the INM is associated with a fibrous, so-called nuclear lamina. Among the NETs, the Sun-domain proteins in the INM (e.g. Sun1) and KASH-domain proteins in the ONM (e.g. nesprin) have to be highlighted, as they form the so-called LINC complex, which spans the whole nuclear envelope (Lee and Burke, 2018...

show abstract

Proximity‐dependent labeling methods for proteomic profiling in living cells

Chen

Perrimon

2017

WIREs Developmental Biology

View full text Add to dashboard Cite

Characterizing the proteome composition of organelles and subcellular regions of living cells can facilitate the understanding of cellular organization as well as protein interactome networks. Proximity labeling-based methods coupled with mass spectrometry (MS) offer a high-throughput approach for systematic analysis of spatially restricted proteomes. Proximity labeling utilizes enzymes that generate reactive radicals to covalently tag neighboring proteins with biotin. The biotinylated endogenous proteins can then be isolated for further analysis by MS. To analyze protein–protein interactions or identify components that localize to discrete subcellular compartments, spatial expression is achieved by fusing the enzyme to specific proteins or signal peptides that target to particular subcellular regions. Although these technologies have only been introduced recently, they have already provided deep insights into a wide range of biological processes. Here, we describe and compare current methods of proximity labeling as well as their applications. As each method has its own unique features, the goal of this review is to describe how different proximity labeling methods can be used to answer different biological questions.

show abstract

Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

Cited by 18 publications

References 53 publications

Meet the neighbors: Mapping local protein interactomes by proximity‐dependent labeling with BioID

Meet the neighbors: Mapping local protein interactomes by proximity‐dependent labeling with BioID

Nuclear envelope organization in Dictyostelium discoideum

Proximity‐dependent labeling methods for proteomic profiling in living cells

Contact Info

Product

Resources

About