Direct membrane protein–DNA interactions required early in nuclear envelope assembly

Among the earliest events in postmitotic nuclear envelope (NE) assembly are the interactions between chromatin and the membranes that will fuse to form the NE. It has been proposed that interactions between integral NE proteins and chromatin proteins mediate initial membrane recruitment to chromatin. We show that several transmembrane NE proteins bind to DNA directly and that NE membrane proteins as a class are enriched in long, basic domains that potentially bind DNA. Membrane fractions that are essential for NE formation are shown to bind directly to protein-free DNA, and our data suggest that these interactions are critical for early steps in NE assembly.

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Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase

BMC Biology ◽

10.1186/s12915-021-01048-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Cheng-Wen He ◽

Xue-Fei Cui ◽

Shao-Jie Ma ◽

Qin Xu ◽

Yan-Peng Ran ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Stationary Phase ◽

Molecular Mechanisms ◽

Multivesicular Body ◽

Degradation Process ◽

Vacuolar Membrane ◽

Yeast Cells ◽

Membrane Structures ◽

Membrane Recruitment

Abstract Background The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. Results Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. Conclusions Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

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Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0199 ◽

2011 ◽

Vol 22 (18) ◽

pp. 3306-3317 ◽

Cited By ~ 32

Author(s):

Li-Chuan Tseng ◽

Rey-Huei Chen

Keyword(s):

Membrane Protein ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Chromatin Binding ◽

Temporal Control ◽

Lamin B ◽

Lamin B Receptor ◽

Nuclear Envelope Assembly ◽

Nuclear Membrane Protein

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Here, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contributes to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

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Factors promoting nuclear envelope assembly independent of the canonical ESCRT pathway

The Journal of Cell Biology ◽

10.1083/jcb.201908232 ◽

2020 ◽

Vol 219 (6) ◽

Cited By ~ 10

Author(s):

I-Ju Lee ◽

Ema Stokasimov ◽

Nathaniel Dempsey ◽

Joseph M. Varberg ◽

Etai Jacob ◽

...

Keyword(s):

Fatty Acids ◽

Membrane Protein ◽

Nuclear Envelope ◽

Unsaturated Fatty Acids ◽

Genetic Screen ◽

Prior Work ◽

General Role ◽

Alternate Pathway ◽

Nuclear Envelope Assembly ◽

Inner Nuclear Membrane Protein

The nuclear envelope (NE) undergoes dynamic remodeling to maintain NE integrity, a process involving the inner nuclear membrane protein LEM2 recruiting CHMP7/Cmp7 and then ESCRT-III. However, prior work has hinted at CHMP7/ESCRT-independent mechanisms. To identify such mechanisms, we studied NE assembly in Schizosaccharomyces japonicus, a fission yeast that undergoes partial mitotic NE breakdown and reassembly. S. japonicus cells lacking Cmp7 have compromised NE sealing after mitosis but are viable. A genetic screen identified mutations that promote NE integrity in cmp7Δ cells. Unexpectedly, loss of Lem2 or its interacting partner Nur1 suppressed cmp7Δ defects. In the absence of Cmp7, Lem2 formed aggregates that appear to interfere with ESCRT-independent NE sealing. A gain-of-function mutation implicated a membrane and ESCRT-III regulator, Alx1, in this alternate pathway. Additional results suggest a potentially general role for unsaturated fatty acids in NE integrity. These findings establish the existence of mechanisms for NE sealing independent of the canonical ESCRT pathway.

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MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells

Cell Reports ◽

10.1016/j.celrep.2018.11.027 ◽

2018 ◽

Vol 25 (10) ◽

pp. 2891-2903.e5 ◽

Cited By ~ 6

Author(s):

Michal Sobecki ◽

Charbel Souaid ◽

Jocelyne Boulay ◽

Vincent Guerineau ◽

Daan Noordermeer ◽

...

Keyword(s):

Nuclear Envelope ◽

Human Cells ◽

Dna Interactions ◽

Contact Sites ◽

Protein Dna Interactions

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A Visual Screen of a Gfp-Fusion Library Identifies a New Type of Nuclear Envelope Membrane Protein

The Journal of Cell Biology ◽

10.1083/jcb.146.1.29 ◽

1999 ◽

Vol 146 (1) ◽

pp. 29-44 ◽

Cited By ~ 128

Author(s):

Melissa M. Rolls ◽

Pascal A. Stein ◽

Stephen S. Taylor ◽

Edward Ha ◽

Frank McKeon ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Nuclear Envelope ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Physical Separation ◽

Culture Cells ◽

New Type ◽

Green Fluorescent ◽

Membrane Components

The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.

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TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

Biochemical Journal ◽

10.1042/bcj20190359 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3241-3260

Author(s):

Sindhu Wisesa ◽

Yasunori Yamamoto ◽

Toshiaki Sakisaka

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Mammalian Cells ◽

Coiled Coil ◽

Transmembrane Domains ◽

Domain Family ◽

Coiled Coil Domain ◽

U2os Cells ◽

Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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