John McCullough

Membrane Fission Reactions of the Mammalian ESCRT Pathway

The endosomal sorting complexes required for transport (ESCRT) pathway was initially defined in yeast genetic screens that identified the factors necessary to sort membrane proteins into intraluminal endosomal vesicles. Subsequent studies have revealed that the mammalian ESCRT pathway also functions in a series of other key cellular processes, including formation of extracellular microvesicles, enveloped virus budding, and the abscission stage of cytokinesis. The core ESCRT machinery comprises Bro1 family proteins and ESCRT-I, ESCRT-II, ESCRT-III, and VPS4 complexes. Site-specific adaptors recruit these soluble factors to assemble on different cellular membranes, where they carry out membrane fission reactions. ESCRT-III proteins form filaments that draw membranes together from the cytoplasmic face, and mechanistic models have been advanced to explain how ESCRT-III filaments and the VPS4 ATPase can work together to catalyze membrane fission.

ESCRT-III Protein Requirements for HIV-1 Budding

Two early-acting components of the cellular ESCRT pathway, ESCRT-I and ALIX, participate directly in HIV-1 budding. The membrane fission activities of ESCRT-III subunits are also presumably required, but humans express 11 different CHMP/ESCRT-III proteins whose functional contributions are not yet clear. We therefore depleted cells of each of the different CHMP proteins and protein families and examined the effects on HIV-1 budding. Virus release was profoundly inhibited by codepletion of either CHMP2 or CHMP4 family members, resulting in ≥100-fold titer reductions. CHMP2A and CHMP4B proteins bound one another, and this interaction was required for budding. By contrast, virus release was reduced only modestly by depletion of CHMP3 and CHMP1 proteins (2- to 8-fold titer reductions) and was unaffected by depletion of other human ESCRT-III proteins. HIV-1 budding therefore requires only a subset of the known human ESCRT-III proteins, with the CHMP2 and CHMP4 families playing key functional roles.

ALIX-CHMP4 interactions in the human ESCRT pathway.

The ESCRT pathway facilitates membrane fission events during enveloped virus budding, multivesicular body formation, and cytokinesis. To promote HIV budding and cytokinesis, the ALIX protein must bind and recruit CHMP4 subunits of the ESCRT-III complex, which in turn participate in essential membrane remodeling functions. Here, we report that the Bro1 domain of ALIX binds specifically to C-terminal residues of the human CHMP4 proteins (CHMP4A-C). Crystal structures of the complexes reveal that the CHMP4 C-terminal peptides form amphipathic helices that bind across the conserved concave surface of ALIXBro1. ALIX-dependent HIV-1 budding is blocked by mutations in exposed ALIXBro1 residues that help contribute to the binding sites for three essential hydrophobic residues that are displayed on one side of the CHMP4 recognition helix (M/L/IxxLxxW). The homologous CHMP1–3 classes of ESCRT-III proteins also have C-terminal amphipathic helices, but, in those cases, the three hydrophobic residues are arrayed with L/I/MxxxLxxL spacing. Thus, the distinct patterns of hydrophobic residues provide a “code” that allows the different ESCRT-III subunits to bind different ESCRT pathway partners, with CHMP1–3 proteins binding MIT domain-containing proteins, such as VPS4 and Vta1/LIP5, and CHMP4 proteins binding Bro1 domain-containing proteins, such as ALIX.

Structural Basis for ESCRT-III Protein Autoinhibition

Endosomal sorting complexes required for transport-III (ESCRT-III) subunits cycle between two states: soluble monomers and higher-order assemblies that bind and remodel membranes during endosomal vesicle formation, midbody abscission and enveloped virus budding. Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member. IST1 and its ESCRT-III binding partner, CHMP1B, both form higher-order helical structures in vitro, and IST1-CHMP1 interactions are required for abscission. The IST1 and CHMP3 structures also reveal that equivalent downstream α5 helices can fold back against the core domains. Mutations within the CHMP3 core–α5 interface stimulate the proteins in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory α5 helix from the core activates ESCRT-III proteins for assembly at membranes.

Structure and membrane remodeling activity of ESCRT-III helical polymers

ESCRTs work in two very different ways The so-called ESCRT proteins are involved in the budding of vesicles into the lumen of endosomes and in virus budding. These reactions involve the formation of a cytoplasm-filled neck that spirals of the ESCRTs help to seal. McCullough et al. now show that ESCRTs can also promote the scission of membrane tubules with the completely opposite topology. ESCRT-III and IST1 ESCRT subunits form spirals on the outside of membrane tubules and so can mediate the budding of tubules and vesicles into the cytosol. Relatively minor structural rearrangements were required to turn ESCRT function on its head. Science, this issue p. 1548 ESCRT-III subunits adopt two different conformations to form external coats that tubulate intracellular membranes. 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.

Electron cryotomography of ESCRT assemblies and dividing Sulfolobus cells suggests that spiraling filaments are involved in membrane scission

ESCRT filaments wrap helically around liposomes and assemble into various helical structures in vitro. Dividing Sulfolobus cells further exhibit a thin, dynamic belt coating division furrows. Together these data suggest that spiraling filaments are involved in membrane scission.

Relatedness and mortality risk during a crisis year: Plymouth colony, 1620–1621

Abstract To test the association of relatedness with risk of death during a demographic crisis, individual relatedness values were compared with mortality histories of the 103 passengers on the 1620 voyage of the Mayflower to Plymouth. Fifty-three (51.5%) died during the first winter due to malnutrition, disease, and lack of preparedness. There was no bias in proportion dying by sex or social class, but children survived in higher proportion (74.2%) than adults (37.5%)(t = 3.75; p t , (0.870; s.d., 0.694) than non-survivors (0.429; s.d., 0.515) (t = 3.61; p s , to decedents, S d , and age; however, only 71.8% of the sample was classified to correct survival category. Percent survivorship and relatedness were associated in simple linear fashion for the entire sample as tested by regression analysis, using either Spearmans or Pearsons methods. Thus, relatedness was directly associated with probability of survival, but the presence of different levels of relatedness for different social and demographic categories suggests that other factors also influence the outcome.

The evolutionary adaptation of the C282Y mutation to culture and climate during the European Neolithic

ABSTRACT Objectives The C282Y allele is the major cause of hemochromatosis as a result of excessive iron absorption. The mutation arose in continental Europe no earlier than 6,000 years ago, coinciding with the arrival of the Neolithic agricultural revolution. Here we hypothesize that this new Neolithic diet, which originated in the sunny warm and dry climates of the Middle East, was carried by migrating farmers into the chilly and damp environments of Europe where iron is a critical micronutrient for effective thermoregulation. We argue that the C282Y allele was an adaptation to this novel environment. Materials and Methods To address our hypothesis, we compiled C282Y allele frequencies, known Neolithic sites in Europe and climatic data on temperature and rainfall for statistical analysis. Results Our findings indicate that the geographic cline for C282Y frequency in Europe increases as average temperatures decrease below 16°C, a critical threshold for thermoregulation, with rainy days intensifying the trend. Discussion The results indicate that the deleterious C282Y allele, responsible for most cases of hemochromatosis, may have evolved as a selective advantage to culture and climate during the European Neolithic. Am J Phys Anthropol 160:86–101, 2016.

Putting a finger in the ring

Two complementary papers demonstrate that the homologous type II transmembrane proteins LAP1 and LULL1 adopt nucleotide-free AAA+ ATPase folds and donate arginine fingers to complete the active sites of Torsin AAA+ ATPases. Activated Torsin complexes appear to function in nuclear and endoplasmic reticulum membrane-remodeling processes, including a nuclear vesiculation pathway that carries large cellular and viral cargoes from the nucleus into the cytoplasm.

Hemochromatosis: Niche Construction and the Genetic Domino Effect in the European Neolithic

ABSTRACT Hereditary hemochromatosis is caused by a potentially lethal recessive gene (HFE, C282Y allele) that increases iron absorption and reaches polymorphic levels in northern European populations. Because persons carrying the allele absorb iron more readily than do noncarriers, it has often been suggested that HFE is an adaptation to anemia. We hypothesize positive selection for HFE began during or after the European Neolithic with the adoption of an iron-deficient high-grain and dairying diet and consequent anemia, a finding confirmed in Neolithic and later European skeletons. HFE frequency compared with rate of lactase persistence in Eurasia yields a positive linear correlation coefficient of 0.86. We suggest this is just one of many mutations that became common after the adoption of agriculture.