Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome.

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.

Heh2/Man1 may be an evolutionarily conserved sensor of NPC assembly state.

Integral membrane proteins of the Lap2-emerin-MAN1 (LEM) family have emerged as important components of the inner nuclear membrane (INM) required for the functional and physical integrity of the nuclear envelope. However, like many INM proteins, there is limited understanding of the biochemical interaction networks that enable LEM protein function. Here, we show that Heh2/Man1 can interact with major scaffold components of the nuclear pore complex (NPC), specifically the inner ring complex (IRC), in evolutionarily distant yeasts. Although an N-terminal domain is required for Heh2 targeting to the INM, we demonstrate that more stable interactions with the NPC are mediated by a C-terminal winged helix (WH) domain, thus decoupling INM targeting and NPC binding. Inhibiting Heh2's interactions with the NPC by deletion of the Heh2 WH domain leads to NPC clustering. Interestingly, Heh2's association with NPCs can also be disrupted by knocking out several outer ring nucleoporins. Thus, Heh2's interaction with NPCs depends on the structural integrity of both major NPC scaffold complexes. We propose a model in which Heh2 acts as a sensor of NPC assembly state, which may be important for NPC quality control mechanisms and the segregation of NPCs during cell division.

Direct binding of ESCRT protein Chm7 to phosphatidic acid-rich membranes at nuclear envelope herniations.

Mechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)-binding capacity in the nuclear envelope (NE)-specific ESCRT, Chm7, in budding yeast. Chm7's interaction with PA-rich membranes is mediated through a conserved hydrophobic stretch of amino acids, which confers recruitment to the NE in a manner that is independent of but required for Chm7's interaction with the LAP2-emerin-MAN1 (LEM) domain protein Heh1 (LEM2). Consistent with the functional importance of PA binding, mutation of this region abrogates recruitment of Chm7 to membranes and abolishes Chm7 function in the context of NE herniations that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that a PA sensor specifically accumulates within these NE herniations. We suggest that local control of PA metabolism is important for ensuring productive NE remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast.

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here, we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells, we observe drastic changes in organellar volume, leading to crowding on the micrometer scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.

Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics.

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC quality control is compromised in aging mitotic cells. Our images of single yeast cells during aging, show that the abundance of several NPC components and NPC assembly factors decreases. Additionally, the single-cell life histories reveal that cells that better maintain those components are longer lived. The presence of herniations at the nuclear envelope of aged cells suggests that misassembled NPCs are accumulated in aged cells. Aged cells show decreased dynamics of transcription factor shuttling and increased nuclear compartmentalization. These functional changes are likely caused by the presence of misassembled NPCs, as we find that two NPC assembly mutants show similar transport phenotypes as aged cells. We conclude that NPC interphase assembly is a major challenge for aging mitotic cells.

An ESCRT-LEM protein surveillance system is poised to directly monitor the nuclear envelope and nuclear transport system.

The integrity of the nuclear membranes coupled to the selective barrier of nuclear pore complexes (NPCs) are essential for the segregation of nucleoplasm and cytoplasm. Mechanical membrane disruption or perturbation to NPC assembly triggers an ESCRT-dependent surveillance system that seals nuclear pores: how these pores are sensed and sealed is ill defined. Using a budding yeast model, we show that the ESCRT Chm7 and the integral inner nuclear membrane (INM) protein Heh1 are spatially segregated by nuclear transport, with Chm7 being actively exported by Xpo1/Crm1. Thus, the exposure of the INM triggers surveillance with Heh1 locally activating Chm7. Sites of Chm7 hyperactivation show fenestrated sheets at the INM and potential membrane delivery at sites of nuclear envelope herniation. Our data suggest that perturbation to the nuclear envelope barrier would lead to local nuclear membrane remodeling to promote membrane sealing. Our findings have implications for disease mechanisms linked to NPC assembly and nuclear envelope integrity.

Fantastic nuclear envelope herniations and where to find them.

Morphological abnormalities of the bounding membranes of the nucleus have long been associated with human diseases from cancer to premature aging to neurodegeneration. Studies over the past few decades support that there are both cell intrinsic and extrinsic factors (e.g. mechanical force) that can lead to nuclear envelope 'herniations', a broad catch-all term that reveals little about the underlying molecular mechanisms that contribute to these morphological defects. While there are many genetic perturbations that could ultimately change nuclear shape, here, we focus on a subset of nuclear envelope herniations that likely arise as a consequence of disrupting physiological nuclear membrane remodeling pathways required to maintain nuclear envelope homeostasis. For example, stalling of the interphase nuclear pore complex (NPC) biogenesis pathway and/or triggering of NPC quality control mechanisms can lead to herniations in budding yeast, which are remarkably similar to those observed in human disease models of early-onset dystonia. By also examining the provenance of nuclear envelope herniations associated with emerging nuclear autophagy and nuclear egress pathways, we will provide a framework to help understand the molecular pathways that contribute to nuclear deformation.

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

The integrity of the nuclear envelope barrier relies on membrane remodeling by the ESCRTs, which seal nuclear envelope holes and contribute to the quality control of nuclear pore complexes (NPCs); whether these processes are mechanistically related remains poorly defined. Here, we show that the ESCRT-II/III chimera, Chm7, is recruited to a nuclear envelope subdomain that expands upon inhibition of NPC assembly and is required for the formation of the storage of improperly assembled NPCs (SINC) compartment. Recruitment to sites of NPC assembly is mediated by its ESCRT-II domain and the LAP2-emerin-MAN1 (LEM) family of integral inner nuclear membrane proteins, Heh1 and Heh2. We establish direct binding between Heh2 and the "open" forms of both Chm7 and the ESCRT-III, Snf7, and between Chm7 and Snf7. Interestingly, Chm7 is required for the viability of yeast strains where double membrane seals have been observed over defective NPCs; deletion of CHM7 in these strains leads to a loss of nuclear compartmentalization suggesting that the sealing of defective NPCs and nuclear envelope ruptures could proceed through similar mechanisms.