IST1 regulates select recycling pathways.

ESCRTs (Endosomal Sorting Complex Required for Transports) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intraluminal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy, we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.

Access of torsinA to the inner nuclear membrane is activity dependent and regulated in the endoplasmic reticulum.

TorsinA (also known as torsin-1A) is a membrane-embedded AAA+ ATPase that has an important role in the nuclear envelope lumen. However, most torsinA is localized in the peripheral endoplasmic reticulum (ER) lumen where it has a slow mobility that is incompatible with free equilibration between ER subdomains. We now find that nuclear-envelope-localized torsinA is present on the inner nuclear membrane (INM) and ask how torsinA reaches this subdomain. The ER system contains two transmembrane proteins, LAP1 and LULL1 (also known as TOR1AIP1 and TOR1AIP2, respectively), that reversibly co-assemble with and activate torsinA. Whereas LAP1 localizes on the INM, we show that LULL1 is in the peripheral ER and does not enter the INM. Paradoxically, interaction between torsinA and LULL1 in the ER targets torsinA to the INM. Native gel electrophoresis reveals torsinA oligomeric complexes that are destabilized by LULL1. Mutations in torsinA or LULL1 that inhibit ATPase activity reduce the access of torsinA to the INM. Furthermore, although LULL1 binds torsinA in the ER lumen, its effect on torsinA localization requires cytosolic-domain-mediated oligomerization. These data suggest that LULL1 oligomerizes to engage and transiently disassemble torsinA oligomers, and is thereby positioned to transduce cytoplasmic signals to the INM through torsinA.

Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum.

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.

IST1 regulates select endosomal recycling pathways.

ESCRTs (Endosomal Sorting Complex Required for Transport) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intralumenal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.

Interaction of torsinA with its major binding partners is impaired by the dystonia-associated DeltaGAG deletion.

Early onset (DYT1) torsion dystonia is a dominantly inherited movement disorder associated with a three-base pair (DeltaGAG) deletion that removes a glutamic acid residue from the protein torsinA. TorsinA is an essential AAA(+) (ATPases associated with a variety of cellular activities) ATPase found in the endoplasmic reticulum and nuclear envelope of higher eukaryotes, but what it does and how changes caused by the DeltaGAG deletion lead to dystonia are not known. Here, we asked how the DYT1 mutation affects association of torsinA with interacting proteins. Using immunoprecipitation and mass spectrometry, we first established that the related transmembrane proteins LULL1 and LAP1 are prominent binding partners for torsinA in U2OS cells. Comparative analysis demonstrates that these two proteins are targeted to the endoplasmic reticulum or nuclear envelope by their divergent N-terminal domains. Binding of torsinA to their C-terminal lumenal domains is stabilized when residues in any one of three motifs implicated in ATP hydrolysis (Walker B, sensor 1, and sensor 2) are mutated. Importantly, the DeltaGAG deletion does not stabilize this binding. Indeed, deleting the DeltaGAG encoded glutamic acid residue from any of the three ATP hydrolysis mutants destabilizes their association with LULL1 and LAP1C, suggesting a possible basis for loss of torsinA function. Impaired interaction of torsinA with LULL1 and/or LAP1 may thus contribute to the development of dystonia.

Triggered recruitment of ESCRT machinery promotes endolysosomal repair.

Endolysosomes can be damaged by diverse materials. Terminally damaged compartments are degraded by lysophagy, but pathways that repair salvageable organelles are poorly understood. Here we found that the endosomal sorting complex required for transport (ESCRT) machinery, known to mediate budding and fission on endolysosomes, also plays an essential role in their repair. ESCRTs were rapidly recruited to acutely injured endolysosomes through a pathway requiring calcium and ESCRT-activating factors that was independent of lysophagy. We used live-cell imaging to demonstrate that ESCRTs responded to small perforations in endolysosomal membranes and enabled compartments to recover from limited damage. Silica crystals that disrupted endolysosomes also triggered ESCRT recruitment. ESCRTs thus provide a defense against endolysosomal damage likely to be relevant in physiological and pathological contexts.

Torsins Are Essential Regulators of Cellular Lipid Metabolism.

Torsins are developmentally essential AAA+ proteins, and mutation of human torsinA causes the neurological disease DYT1 dystonia. They localize in the ER membranes, but their cellular function remains unclear. We now show that dTorsin is required in Drosophila adipose tissue, where it suppresses triglyceride levels, promotes cell growth, and elevates membrane lipid content. We also see that human torsinA at the inner nuclear membrane is associated with membrane expansion and elevated cellular lipid content. Furthermore, the key lipid metabolizing enzyme, lipin, is mislocalized in dTorsin-KO cells, and dTorsin increases levels of the lipin substrate, phosphatidate, and reduces the product, diacylglycerol. Finally, genetic suppression of dLipin rescues dTorsin-KO defects, including adipose cell size, animal growth, and survival. These findings identify that torsins are essential regulators of cellular lipid metabolism and implicate disturbed lipid biology in childhood-onset DYT1 dystonia.

Structure and membrane remodeling activity of ESCRT-III helical polymers.

The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises "open" CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of "closed" IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.

LULL1 retargets TorsinA to the nuclear envelope revealing an activity that is impaired by the DYT1 dystonia mutation.

TorsinA (TorA) is an AAA+ ATPase in the endoplasmic reticulum (ER) lumen that is mutated in early onset DYT1 dystonia. TorA is an essential protein in mice and is thought to function in the nuclear envelope (NE) despite localizing throughout the ER. Here, we report that transient interaction of TorA with the ER membrane protein LULL1 targets TorA to the NE. FRAP and Blue Native PAGE indicate that TorA is a stable, slowly diffusing oligomer in either the absence or presence of LULL1. Increasing LULL1 expression redistributes both wild-type and disease-mutant TorA to the NE, while decreasing LULL1 with shRNAs eliminates intrinsic enrichment of disease-mutant TorA in the NE. When concentrated in the NE, TorA displaces the nuclear membrane proteins Sun2, nesprin-2G, and nesprin-3 while leaving nuclear pores and Sun1 unchanged. Wild-type TorA also induces changes in NE membrane structure. Because SUN proteins interact with nesprins to connect nucleus and cytoskeleton, these effects suggest a new role for TorA in modulating complexes that traverse the NE. Importantly, once concentrated in the NE, disease-mutant TorA displaces Sun2 with reduced efficiency and does not change NE membrane structure. Together, our data suggest that LULL1 regulates the distribution and activity of TorA within the ER and NE lumen and reveal functional defects in the mutant protein responsible for DYT1 dystonia.

Effects of genetic variations in the dystonia protein torsinA: identification of polymorphism at residue 216 as protein modifier.

Four naturally occurring sequence variations have been found in the coding region of the DYT1 gene encoding torsinA. One of these, a 3 bp (DeltaGAG) deletion, underlies dominantly inherited cases of early-onset torsion dystonia. Others, including a single nucleotide polymorphism that replaces aspartic acid (D) at residue 216 with histidine (H) in 12% of normal alleles and two other rare deletions, have not been clearly associated with disease. To gain insight into how these sequence variations affect torsinA, we used the structure of the related protein ClpB to provide a model of torsinA's AAA+ domain. Motifs important for ATP hydrolysis-sensor 1 and sensor 2-were identified, mutagenized and used to validate predictions of this model. Inspection revealed that the DeltaGAG deletion associated with dystonia removes one residue from an alpha-helix in the C-terminal portion of the AAA+ domain. The resulting distortion in torsinA structure may underlie this mutant's known tendency to produce ER-derived inclusions as well as its proposed loss of function. The D/H polymorphism at residue 216 falls in the N-terminal portion of the AAA+ domain near the sensor 1 motif. Surprisingly, cells expressing torsinA with the polymorphic histidine developed inclusions similar to those associated with DeltaGAG-torsinA, indicating that this change may also affect torsinA structure. Introducing H216 into DeltaGAG-torsinA reduced its tendency to form inclusions, suggesting that the two changes offset each other. Our findings point to a structural basis for the defects associated with the disease-linked DeltaGAG deletion in torsinA. They also suggest possible connections between the allelic polymorphism at residue 216 and the penetrance of DYT1 dystonia, as well as a possible role for this polymorphism in related disease states.

Interaction of the mammalian endosomal sorting complex required for transport (ESCRT) III protein hSnf7-1 with itself, membranes, and the AAA+ ATPase SKD1.

SKD1/VPS4B is an AAA+ (ATPase associated with a variety of cellular activities) protein involved in multivesicular body (MVB) biogenesis. In this study, we show that the impairment in MVB biogenesis caused by the ATP hydrolysis-deficient mutant SKD1(E235Q) is accompanied by assembly of a large detergent-insoluble protein complex that includes normally soluble endogenous components of mammalian endosomal sorting complex required for transport (ESCRT) I and ESCRT-III complexes. Membrane-bound ESCRT-III complex has been proposed to be the substrate that recruits SKD1 to nascent MVBs. To explore this relationship, we studied interactions among the human ESCRT-III components hSnf7-1 and hVps24, membranes, and SKD1. We found that a significant portion of overexpressed hSnf7-1 associated with membranes where it formed a large protein complex that recruited SKD1 and perturbed normal MVB biogenesis. Overexpressed hVps24 also associated with membranes and perturbed endosome structure but only when fused to green fluorescent protein. Domain analysis revealed that the basic N-terminal half of hSnf7-1 localized to membranes and formed detergent-resistant polymers, some of which looked like filopodia extending into the lumen of swollen endosomes or out from the plasma membrane. The C-terminal acidic half of hSnf7-1 did not associate with membranes and was required for interaction of hSnf7-1 with SKD1. Together with earlier studies, our work suggests that a variety of ESCRT-III-containing polymers can assemble on membranes and recruit SKD1 during formation of the MVB.

TorsinA in the nuclear envelope.

Early-onset torsion dystonia, a CNS-based movement disorder, is usually associated with a single amino acid deletion (Delta E302/303) in the protein torsinA. TorsinA is an AAA+ ATPase in the endoplasmic reticulum, but what it does is unknown. Here, we use torsinA mutants with defects in ATP hydrolysis (E171Q, ATP-bound) and ATP binding (K108A, ATP-free) to probe torsinA's normal cellular function. Surprisingly, ATP-bound torsinA is recruited to the nuclear envelope (NE) of transfected cells, where it alters connections between inner and outer nuclear membranes. In contrast, ATP-free torsinA is diffusely distributed throughout the endoplasmic reticulum and has no effect on the NE. Among AAA+ ATPases, affinity for substrates is high in the ATP-bound and low in the ATP-free state, leading us to propose that component(s) of the NE may be substrates for torsinA. We also find that the disease-promoting Delta E302/303 mutant is in the NE, and that this relocalization, as well as the mutant's previously described ability to induce membranous inclusions, is eliminated by the K108A ATP-binding mutation. These results suggest that changes in interactions involving torsinA in the NE could be important for the pathogenesis of dystonia and point to torsinA and related proteins as a class of ATPases that may operate in the NE.