LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

Abstract

ESCRT-III proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In S. pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disc periphery during this window of cell division, and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear sitespecific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/049312 doi: bioRxiv preprint first posted online Apr. 19, 2016; 3 SignificanceThe molecular mechanism for sealing newly formed nuclear envelopes was unclear until the recent discovery that ESCRT-III proteins mediate this process. CHMP7, in particular, was identified as an early-acting factor that recruits other ESCRT-III proteins to the nuclear envelope.A fundamental aspect of the varied roles of ESCRT factors is their recruitment by site-specific adaptors, yet the central question of how the ESCRT machinery is targeted to nuclear membranes has remained outstanding. Our study identifies the inner nuclear membrane protein LEM2 as a key, conserved factor that recruits CHMP7 and downstream ESCRT-III proteins to breaches in the nuclear envelope.

Membrane binding by CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation

“…Recent reports describing the association of CHMP7's C terminus with LEM family proteins [21] and that LEMD2 depletion impairs ESCRT-III assembly at this organelle [22] indicate that these also may be candidates that regulate the enrichment of CHMP7 at sites of annular fusion.…”

“…Recent reports describing the association of CHMP7's C terminus with LEM family proteins [21] and that LEMD2 depletion impairs ESCRT-III assembly at this organelle [22] indicate that these also may be candidates that regulate the enrichment of CHMP7 at sites of annular fusion. While CHMP7's membrane binding was curvature insensitive, a geometric constraint of the narrow-radius hole that is to be closed also may restrict Figures S3B-S3D) were mutated to alanine.…”

“…In the absence of membranebound CHMP7, ESCRT-III cannot assemble at the NE. CHMP7 NT was not stabilized at the reforming NE, suggesting that subsequent engagement of ESCRT-III [11] (and ESCRT-III-binding partners such as Spastin [3] or UFD1L [2]) by the C terminus of CHMP7 provides a stabilizing cue.Recent reports describing the association of CHMP7's C terminus with LEM family proteins [21] and that LEMD2 depletion impairs ESCRT-III assembly at this organelle [22] indicate that these also may be candidates that regulate the enrichment of CHMP7 at sites of annular fusion. …”

Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing

“…Our observation that SINC formation (and the aggravating synthetic growth delays of vps4 Δ pom152 Δ cells) is dependent on CHM7 (Fig A), supports such a model of a toxic gain‐of‐function of Chm7 when it is not properly regulated. Interestingly, in a sister study in fission yeast, deletion of the CHM7 orthologue CMP7 also suppressed the growth delays and NE morphological abnormalities observed in fission yeast vps4 Δ cells (as does LEM2/HEH1 deletion) suggesting the broad conservation of these functional interaction networks (Gu et al , ). We speculate that the over activation of a Chm7‐dependent NE pore closure machinery could lead to the inappropriate sealing of nascent NE pores essential for normal NPC assembly, which might explain why the SINC is enriched for newly synthesized NPCs without their full complement of nups (Webster et al , ).…”

Poor old pores—The challenge of making and maintaining nuclear pore complexes in aging

“…Interference with this quality control mechanism results in the accumulation of misassembled NPCs . The proteins that guard NPC assembly in baker's yeast are conserved in higher eukaryotes where Chmp7 and the ESCRT‐III system additionally have the important function of resealing the NE at the end of mitosis . In baker's yeast, several proteins that assist and control the quality of NPC assembly decrease in abundance during aging, and there are indications that aged yeast cells experience problems with NPC assembly (more details in Box 3).…”