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

Abstract

Mechanisms that turnover 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 shows 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 an integral INM protein. Interestingly, correlative light and electron tomography 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.

Nuclear envelope budding and its cellular functions

“…More recently, different molecular markers have also been used to study NEB in light microscopy. These markers include nuclear localization signals fused to a fluorophore, other proteins found at the nuclear surface, or viral nuclear egress proteins [ 28 , 30 , 34 , 61–64 ]. However, only a few studies have thoroughly correlated their light microscopy markers, for example, nuclear lamins in D. melanogaster [ 28 ], Atg39 in S. cerevisiae [ 30 ], or viral proteins [ 63 , 65 , 66 ] with NEB events within the perinuclear space using electron microscopy, which is needed to definitively identify NEB events.…”

“…These markers include nuclear localization signals fused to a fluorophore, other proteins found at the nuclear surface, or viral nuclear egress proteins [ 28 , 30 , 34 , 61–64 ]. However, only a few studies have thoroughly correlated their light microscopy markers, for example, nuclear lamins in D. melanogaster [ 28 ], Atg39 in S. cerevisiae [ 30 ], or viral proteins [ 63 , 65 , 66 ] with NEB events within the perinuclear space using electron microscopy, which is needed to definitively identify NEB events. Optimizing markers to study NEB and the use of super-resolution light microscopy will be essential to progress and provide a mechanistic understanding of this exciting research field.…”

“…Recent evidence suggests that NEB-like mechanisms also contribute to the turnover of damaged INM components via a selective mode of autophagy termed nucleophagy [ 30 , 107 ] (reviewed in [ 106 ]). In yeast, autophagy can be induced when cells are starved of nitrogen or treated with the drug rapamycin.…”

“…In yeast, autophagy can be induced when cells are starved of nitrogen or treated with the drug rapamycin. Both treatments resulted in nuclear envelope protrusions [ 107 ] and NEB [ 30 ] followed by vacuolar removal of INM and nuclear cargo ( Figure 3c ). The autophagy receptor Atg39 is a transmembrane protein that spans the ONM [ 108 ] and connects to the luminal leaflet of the INM via membrane-binding amphipathic helices [ 30 , 107 ].…”

“…More recently, nuclear envelope budding (NEB) was proposed to be an endogenous mechanism of nuclear transport and shown to be present in all organisms investigated under normal growth conditions [ 27 ], suggesting that NEB-like mechanisms were likely co-opted by herpesviruses for nuclear egress. Recent studies have suggested that NEB can serve to transport ribonucleoproteins (RNPs) [ 28 , 29 ], protein aggregates [ 27 ] and inner nuclear membrane components [ 30 ] (also reviewed in [ 31 ]). Discovery and characterization of NEB has until now occurred via ultrastructural analysis.…”