Biology subjects

Levine, T. P.

Publications and source records attributed to Levine, T. P..

8 recordsLinked to original sources

bioRxiv2023

The punctate localisation of the yeast sterol transporter Ysp2p is determined by three dimerisation interfaces in its C-terminus

Sterol lipids traffic between intracellular compartments by vesicular and non-vesicular routes. Sterol traffic from the plasma membrane to the endoplasmic reticulum (ER), so-called retrograde traffic, particularly depends on a non-vesicular mechanism, being transported by the ubiquitous family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs, also called GRAMD1/Asters in humans, VASt in plants). LAMs are similar to many lipid transfer proteins in that they localise to membrane contact sites and carry lipids between two organelles. In yeast, the major LAM active at ER-plasma membrane contact sites is Ysp2p, which has a uniquely punctate distribution in the cortical ER. Here, we have comprehensively dissected how Ysp2p achieves its distinctive punctate localisation. We show that the PHGRAM domain of Ysp2p has membrane binding properties similar to its human counterpart GRAMD1B, but that this is not important for punctate localisation of Ysp2p. Instead, all regions necessary for the punctate localisation of Ysp2p at membrane contacts are present in [~]200 residues at the C-terminus of Ysp2p, with a critical region being a small y-sheet that we predict homodimerises. We also study the role of punctate localisation of Ysp2 in its function in retrograde sterol traffic, and show that function does not require the punctate localisation, but instead requires a polybasic region adjacent to the sterol transfer domain. Finally, to investigate the interaction of the polybasic region with the plasma membrane, we examine contacts populated by the Ysp2 C-terminus by electron tomography, and find that they consist of generic cortical ER.

cell biology↗

bioRxiv2022

Sequence analysis and structural predictions of lipid transfer bridges in the repeating beta groove (RBG) superfamily reveals past and present domain variations affecting form, function and interactions of VPS13, ATG2, SHIP164, Hobbit and Tweek

Lipid transfer between organelles requires proteins that shield the hydrophobic portions of lipids as they cross the cytoplasm. In the last decade a new structural form of lipid transfer protein (LTP) has been found: long hydrophobic grooves made of beta-sheet that bridge between organelles at membrane contact sites. Eukaryotes have five families of bridge-like LTPs: VPS13, ATG2, SHIP164, Hobbit and Tweek. These are unified into a single superfamily through their bridges being composed of just one domain, called the repeating beta groove (RBG) domain, which builds into rod shaped multimers with a hydrophobic-lined groove and hydrophilic exterior. Here, sequences and predicted structures of the RBG superfamily were analyzed in depth. Phylogenetics showed that the last eukaryotic common ancestor contained all five RBG proteins, with duplicate VPS13s. These appear to have arisen in even earlier ancestors from shorter forms with 4 RBG domains. The extreme ends of most RBG proteins have amphipathic helices that might be an adaptation for direct or indirect bilayer interaction, although this has yet to be tested. The one exception to this is the C-terminus of SHIP164, which instead has a coiled-coil. Finally, almost the entire length of the exterior surfaces of the RBG bridges are shown to have conserved residues, indicating sites for partner interactions almost all of which are unknown. These findings can inform future cell biological and biochemical experiments.

bioinformatics↗

bioRxiv2021

Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution

Actively maintained close appositions, or contact sites, between organelle membranes, enable the efficient transfer of biomolecules between the various cellular compartments. Several such sites have been described together with their tethering machinery. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize the proteome of contact sites and support the discovery of new tethers and functional molecules, we established a high throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, two of which have never been studied before, on the background of 1165 strains expressing a mCherry-tagged yeast protein that have a cellular punctate distribution (a hallmark of contact sites). By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified one set of hits as previously unrecognized homologs to Vps13 and Atg2. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell.

cell biology↗

bioRxiv2021

TMEM106B in humans and Vac7 and Tag1 in yeast are predicted to be lipid transfer proteins

TMEM106B is an integral membrane protein of late endosomes and lysosomes involved in neuronal function, its over-expression being associated with familial frontotemporal lobar degeneration, and under-expression linked to hypomyelination. It has also been identified in multiple screens for host proteins required for productive SARS-CoV2 infection. Because standard approaches to understand TMEM106B at the sequence level find no homology to other proteins, it has remained a protein of unknown function. Here, the standard tool PSI-BLAST was used in a non-standard way to show that the lumenal portion of TMEM106B is a member of the LEA-2 domain superfamily. The non-standard tools (HMMER, HHpred and trRosetta) extended this to predict two yeast LEA-2 proteins in the lumenal domains of the degradative vacuole, equivalent to the lysosome: one in Vac7, a regulator of PI(3,5)P2 production, and three in Tag1 which signals to terminate autophagy. Further analysis of previously unreported LEA-2 structures indicated that LEA-2 domains have a long, conserved lipid binding groove. This implies that TMEM106B, Vac7 and Tag1 may all be lipid transfer proteins in the lumen of late endocytic organelles.

bioinformatics↗

bioRxiv2021

Fungal Ice2p has remote homology to SERINCs, restriction factors for HIV and other viruses

Ice2p is an integral endoplasmic reticulum (ER) membrane protein in budding yeast S. cerevisiae named ICE because it is required for Inheritance of Cortical ER. Ice2p has also been reported to be involved in an ER metabolic branch-point that regulates the flux of lipid either to be stored in lipid droplets or to be used as membrane components. Alternately, Ice2p has been proposed to act as a tether that physically bridges the ER at contact sites with both lipid droplets and the plasma membrane via a long loop on the proteins cytoplasmic face that contains multiple predicted amphipathic helices. Here we carried out a bioinformatic analysis to increase understanding of Ice2p. Firstly, regarding topology, we found that diverse members of the fungal Ice2 family have ten transmembrane helices, which places the long loop on the exofacial face of Ice2p, where it cannot form inter-organelle bridges. Secondly, we identified Ice2 as a full-length homologue of SERINC (serine incorporator), a family of proteins with ten transmembrane helices found universally in eukaryotes. Since SERINCs are potent restriction factors for HIV and other viruses, study of Ice2p may reveal functions or mechanisms that shed light on viral restriction by SERINCs.

bioinformatics↗

bioRxiv2020

Evolution and insights into the structure and function of the DedA superfamily containing TMEM41B and VMP1

TMEM41B and VMP1 are endoplasmic reticulum (ER)-localizing multi-spanning membrane proteins required for ER-related cellular processes such as autophagosome formation, lipid droplet homeostasis, and lipoprotein secretion in eukaryotes. Both proteins have a VTT domain, which is similar to the DedA domain found in bacterial DedA family proteins. However, the molecular function and structure of the DedA and VTT domains (collectively referred to as DedA domains) and the evolutionary relationships among the DedA domain-containing proteins are largely unknown. Here, we conduct remote homology search and identify a new clade consisting mainly of bacterial PF06695 proteins of unknown function. Phylogenetic analysis reveals that the TMEM41, VMP1, DedA, and PF06695 families form a superfamily with a common origin, which we term the DedA superfamily. Coevolution-based structural prediction suggests that the DedA domain contains two reentrant loops that face each other in the membrane. This topology is biochemically verified by the substituted cysteine accessibility method. The predicted structure is topologically similar to that of the substrate-binding region of Na+-coupled glutamate transporter solute carrier 1. A potential ion-coupled transport function of the DedA superfamily proteins is discussed.

cell biology↗

bioRxiv2019

Spermiogenesis in the acoel Symsagittifera roscoffensis: nucleus-plasma membrane contact sites and microtubules.

Symsagittifera roscoffensis is a small marine worm found in the intertidal zone of sandy beaches around the European shores of the Atlantic. S. roscoffensis is a member of the Acoelomorpha, a group of flatworms formerly classified with the Platyhelminthes, but now recognised as Xenacoelomorpha, a separate phylum of disputed affinity. We have used electron microscopy to examine the process of spermiogenesis (the final stage of spermatogenesis) in S. roscoffensis, by which spermatids form highly elongated spermatozoa. Their nuclei are long and thread-like, running most of the cells length and during the process a pair of flagella are fully incorporated into the cell body. Two previously undescribed inter-organelle contact sites form at different stages of spermiogenesis. Strikingly, there is an extensive nucleus-plasma membrane contact site. Golgi-derived granules containing electron-dense filaments line up along the spermatid plasma membrane, undergo a conformational change, and donate material that forms a peri-nuclear layer that cements this contact site. We also show in earlier stage spermatids that the same granules are associated with microtubules, presumably for traffic along the elongating cell. We identify a second spermiogenesis-specific contact site where sheaths engulfing each internalising flagellum contact the nuclear envelope. Finally, detailed studies of the spermatozoon axonemes show that the central keel has varying numbers of microtubules along the length of the cell, and is likely to be a centriole derivative.\n\nSummary sentenceDuring spermiogenesis in the acoel flatworm Symsagittifera roscoffensis, two previously unidentified contact sites contribute to the structure of the mature spermatozoon and the axonemal structures show direct continuity between doublet and dense core microtubules.

cell biology↗

bioRxiv2017

Lipid droplet and peroxisome biogenesis occur at the same ER subdomains.

Nascent lipid droplet (LD) formation occurs in the ER membrane1-4. It is not known whether LD biogenesis occurs stochastically in the ER or at subdomains with unique protein and lipid composition. We previously identified ER subdomains in S. cerevisiae that contain Pex30, a reticulon-like ER-resident membrane protein5. There are ~25 Pex30-containing puncta in the ER per cell. These sites are regions where preperoxisomal vesicles (PPVs) are generated5. Here we show that Pex30 subdomains are also the location where most nascent LDs form. Mature LDs remain associated with Pex30 subdomains and the same Pex30 subdomain can simultaneously associate with a LD and a PPV. Pex30 subdomains become highly enriched in diacylglycerol (DAG) during LD biogenesis, indicating they have a unique lipid composition. We find that in higher eukaryotes multiple C2 domain containing transmembrane protein (MCTP2) is the functional homologue of Pex30; MCTP2 resides in ER subdomains where most nascent LD biogenesis occurs and that are often associated with peroxisomes. Together, these findings indicate that most LDs and PPVs form and remain associated with conserved ER subdomains and suggest a link between LD and peroxisome biogenesis.

cell biology↗