https://www.stokeslabnyu.org/contact daily 0.75 2022-07-04 https://images.squarespace-cdn.com/content/v1/5ce435d6bea1ed00016ba287/1558964652247-5MWXNMFZVOPGE4TY6U2Q/MHeiderich_ReflexionenZwei-05-copy.jpg Contact https://www.stokeslabnyu.org/research daily 0.75 2022-06-20 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655499144894-VCJ1HIYP2M7UXL5R7LFV/micrograph.png Research - Potassium transport in bacteria by KdpFABC An unholy marriage between a channel and a pump https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655500451524-9N7L4RVYP9O72123OMGI/yiip_siteA_padded.png Research - Zinc transport by Cation Diffusion Facilitators A homo-dimer with three distinct Zn-binding sites https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655500102121-6TF6Y40G4COMHXRW3TAK/pin8_active_site.png Research - Auxin transport in plants by PIN proteins An elevator mechanism from an unexpected source https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655500342390-C09VTE0PCNG5A9VAMVFM/ncr1_topology_padded.png Research - Sterol transport by the NPC system The glycocalyx is the barrier for this substrate https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655499919875-RFNPID51E2Y77V4JZL0U/snapwts53seg.png Research - Cardiolipin synthesis in mitochondria The lipid phase promotes activity of tafazzin https://images.squarespace-cdn.com/content/v1/5ce435d6bea1ed00016ba287/1558964652247-5MWXNMFZVOPGE4TY6U2Q/MHeiderich_ReflexionenZwei-05-copy.jpg Research https://www.stokeslabnyu.org/people daily 0.75 2024-04-23 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655600155840-SHQ8UKWM4KV0S6KR286O/Gethin.jpg People https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655600618294-EGWMFJL1R0JM4ME7PWBK/stefan_crop.jpg People 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https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/b3019195-17b0-4bbe-bb78-c558237f64c2/peter_bjork.jpg People https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/e1071bc9-ff9c-4eb6-bd2e-7a490b34d884/patricia_matthiasen.jpg People https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/3eea5c31-4eb2-433c-a25e-e3f39fd732f5/leah_epstein_orig.jpg People https://www.stokeslabnyu.org/publications daily 0.75 2022-07-01 https://images.squarespace-cdn.com/content/v1/5ce435d6bea1ed00016ba287/1558964652247-5MWXNMFZVOPGE4TY6U2Q/MHeiderich_ReflexionenZwei-05-copy.jpg Publications https://www.stokeslabnyu.org/research-copy daily 0.75 2022-07-05 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1657039237885-A5OIV932J39HG2SYB3TZ/1-micrograph.jpeg Research (Copy) - Potassium transport in bacteria by KdpFABC An unholy marriage between a channel and a pump https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1657041695141-LB11NPC4LUBAC39CCK8I/yiip_siteA_padded-alt+copy.jpg Research (Copy) - Zinc transport by Cation Diffusion Facilitators A homo-dimer with three distinct Zn-binding sites https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1657039340652-XH5B3QNJ1ZXK5SWSQT1Y/pin8_active_site.jpeg Research (Copy) - Auxin transport in plants by PIN proteins An elevator mechanism from an unexpected source https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1657039366134-DGPRRP3WI2KCC5RRBWN5/ncr1_topology_padded.png Research (Copy) - Sterol transport by the NPC system The glycocalyx is the barrier for this substrate https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1657039391369-0EWME2L0SDON137YCLLU/snapwts53seg.png Research (Copy) - Cardiolipin synthesis in mitochondria The lipid phase promotes activity of tafazzin https://images.squarespace-cdn.com/content/v1/5ce435d6bea1ed00016ba287/1558964652247-5MWXNMFZVOPGE4TY6U2Q/MHeiderich_ReflexionenZwei-05-copy.jpg Research (Copy) https://www.stokeslabnyu.org/home daily 1.0 2024-05-27 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655397298950-HY99FYY0VA1CX7098HLE/kdp_cartoon_border.png Home Proposed mechanism by which KdpFABC uses the energy from ATP hydrolysis to drive potassium (K+) transport into the cell. Activation of KdpFABC is essential for the survival of bacteria under osmotic stress and when their environment has low levels of potassium (e.g., fresh water) https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655396206202-D89BPHTV2XMDK6HUDYCA/personnel_2015.jpg Home - Our Group Postdocs, grad students, student interns and technicians from all over the world work together in our lab in the Department of Cell Biology at NYU School of Medicine and collaborate with a variety of other labs both locally and internationally https://www.stokeslabnyu.org/home/ncr1 monthly 0.5 2022-06-16 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656696398862-VAC936AQQBG874T6JNNC/ncr1_sterol_physiol.png Home - Sterol transport by Ncr1 in yeast - Sterol transport by the NPC system in yeast Cholesterol is an essential element of cellular membranes and is a precursor for a variety of sterols, some of which act as hormones. Cholesterol is synthesized in the smooth endoplasmic reticulum, but is also obtained from dietary sources and recovered from lipid droplets. which serve as a storage compartment. The lysosome plays a pivotal role in trafficking of sterols with the NPC system ultimately transporting these amphiphilic molecules through the glycocalyx and depositing them in the membrane. The NPC system consists of two components and is named after Niemann-Pick disease resulting from cholesterol deficiency. NPC2 is a soluble carrier protein that binds cholesterol within the lysosome and transfers it to NPC1, which moves it into the membrane. In collaboration with the laboratory of Bjørn Pedersen at Aarhus University, we study the yeast homolog of NPC1, NCR1. In yeast, the primary sterol is ergosterol and the vacuole plays a role analogous to the lysosome. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656698175597-VKKOB9RF1QNNO7F0DHA0/ncr1_RND_topology.png Home - Sterol transport by Ncr1 in yeast - NCR1 has two copies of the RND fold The Resistance-Nodulation-Division (RND) family includes a variety of membrane transporters, the best known of which is the multidrug resistance protein AcrB. The family is characterized by the RND fold consisting of 5+1 transmembrane helices separated by an extracellular “sandwich” domain. The proteins typically oligomerize with AcrB forming a homotrimer. NCR1 and its human homolog NPC1 consist of two tandem copies of the RND fold, thus displaying pseudo-symmetry within a single polypeptide. In addition, an N-terminal domain (NTD) is present in the lumen and tethered to the membrane by an additional transmembrane helix (TM1). The NTD initially receives sterol the soluble component NPC2. Although the RND pseudo repeats are firmly associated with one another, the NTD and TM1 appear to be more loosely bound to the core of the protein. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656698340441-HGD6I3C3FNTFDF3VIHV1/ncr1_structure_with_tunnel_inset.png Home - Sterol transport by Ncr1 in yeast - NCR1 structure reveals a hydrophobic tunnel for sterol transport The Pedersen lab has solved structures of NCR1 and NPC2, the former revealing the juxtaposition of the RND domains and the NTD and the latter shedding light on the transfer process. The structure of NCR1 also reveals a continuous tunnel that runs from the sterol binding site in the NTD, between the pseudo-symmetric lumenal domains (MLD and CTD) and into the lumenal leaflet of the bilayer. This tunnel - shown in the inset on the lower left - is postulated to provide a pathway for carrying sterol through the glycocalyx which is presumed to line the lumenal surface of the vacuole. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656699640778-33EWTXG9N31IOBLEN6FG/ncr1_mech_model.png Home - Sterol transport by Ncr1 in yeast - Mechanistic model for sterol transport Current structures from the Pedersen laboratory and others give rise to a model for sterol transport by the NPC system. The cycle starts by loading NPC2 with sterol within the lumen of the vacuole or lysosome. NPC2 then transfers the sterol to the NTD of NCR1 (or NPC1 in lysosomes). After dissociation of this complex, the NTD transfers the sterol into the tunnel between MLD and CTD domains, where it passes across the glycocalyx to reach the lumenal leaflet of the membrane. The sterol is then released from the so-called sterol sensing domain composed by the first set of transmembrane helices (TM2-7). https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/8ff21a51-0f9e-4758-a016-408e7b3063d2/Figure4_v21_acidpair.png Home - Sterol transport by Ncr1 in yeast - Acidic pair may hold the key for energy coupling A conserved pair of acidic residues is seen at the interface between transmembrane domains of many RND proteins (D631 & E1068 for Ncr1). This pair is generally flanked by a basic residue, in this case His1072. Although proton coupling is well established for multidrug resistance RND transporters, the driving force for sterol transport is not yet established. Recent cryo-EM structures of NCR1 at different pHs shows that the H-bonding of this acidic pair is switched from the His to a Lys and that associated allosteric movements of the RND domains are associated with movement of sterol through the tunnel. This led to a proposed mechanism by which pH driven conformational change gives rise to sterol transport across the glycocalyx. https://www.stokeslabnyu.org/home/yiip monthly 0.5 2022-06-16 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655754719386-H3BM1UYN23YG164IX9S3/Znt_family.png Home - Zinc transport by Cation Diffusion Facilitators - Zinc transport by Cation Diffusion Facilitators Ten percent of proteins are estimated to bind zinc. In particular, zinc plays essential roles as cofactor for catalytic and redox enzymes as well as a structural element for transcription factors. Zinc is found at high levels in certain organelles, such as secretory vesicles associated with insulin, glutamanergic synapses, mother’s milk, and semen. The immune response is strongly dependent on zinc and so-called zincosomes produce a dramatic zinc “spark” that prevents polyspermy during fertilization of vertebrate eggs. Although the total concentration of Zn is > 0.1 mM, essentially all of it is bound either by protein or cytoplasmic chelating agents. The distribution of Zn within the cell is controlled by a network of transporters from several different protein families. In humans, export of Zn from the cytoplasm is the responsibility of proteins from the Cation Diffusion Facilitator family, named Znt1-10. They are accompanied by the ZIP family, generally responsible for import of Zn into the cell, and by ABC transporters and P-type pumps in some situations. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655764114053-5IFTHYOCIMEXVT6UI9HF/yiip_overview.png Home - Zinc transport by Cation Diffusion Facilitators - Bacterial Zn transporter YiiP YiiP a bacterial member of the Cation Diffusion Facilitator family. We study the protein from Shewanella oneidensis, which is highly homologous to the protein from E. coli. Like most members of the family, YiiP forms a homodimer with six transmembrane helices and a C-terminal domain in the cytoplasm. YiiP operates as a Zn/H antiporter, meaning that it exchanges Zn(2+) ions with protons (H+) and therefore can use the proton-motive force as an energy source to drive transport. YiiP has conserved Zn-binding sites (pink spheres) within the transmembrane domain (site A) and the C-terminal domain (site C). In addition, YiiP has a non-conserved site in a loop between transmembrane helices 2 and 3 (site B). One of our main interests is to understand the role of these individual sites in transport. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655844584866-P31OYW6U8L0NR0Q70BDD/xlink_summary.png Home - Zinc transport by Cation Diffusion Facilitators - Crosslinking to probe conformational change Cryo-EM structures both in lipid membranes and in detergent micelles have compact transmembrane domains (blue structure), whereas the initial X-ray structure from the Fu laboratory (pink structure) showed transmembrane domains in a splayed conformation. This comparison suggested that transport might be accompanied by a scissoring movement. To test this idea, we engineered cysteine residues at four sites along the dimer interface (yellow spheres) and used copper to induce crosslinking of the dimer (band marked “D” on the gel comparing a Cys-free construct, C190A, and four different mutants) . Assays of transport indicate that the crosslinking had no effect, suggesting that this conformational change is not essential for transport. The D51A and K79D mutations inactivate YiiP and served as negative controls for transport. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655766780473-R1TK3E9UIIVI9RIM4I9M/fab4_modelcolored_front.jpg Home - Zinc transport by Cation Diffusion Facilitators https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655849028012-OCB0GQLS314WMMN4JH82/D51A_summary.png Home - Zinc transport by Cation Diffusion Facilitators https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655849060004-XH2K6WFYB8N213QPGXNI/D70A_summary.png Home - Zinc transport by Cation Diffusion Facilitators https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655849179640-MW9S1CI3NCIVIEVA9TEH/D287A-H263A_summary.png Home - Zinc transport by Cation Diffusion Facilitators https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656957759626-1L54CGHJVP3DR4O280LI/type1a_5vrf.png Home - Zinc transport by Cation Diffusion Facilitators https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656958018527-GS1WR5WEIW9773VRIE8Q/tubular_xtals.jpg Home - Zinc transport by Cation Diffusion Facilitators https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656958041049-6TOM7IM8S00ZB4PBYMCC/yiip_SPA_image.jpg Home - Zinc transport by Cation Diffusion Facilitators https://www.stokeslabnyu.org/home/pin monthly 0.5 2022-06-17 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655920460033-UJ3621PF1VVAJ9HGNTNI/auxin_intro.jpg Home - Auxin transport by PIN8 in plants - Auxin transport in plants by PIN8 Auxin is a key regulator of plant growth and development. It is responsible for phototropism, which is the ability of plants to direct growth towards a light source. In addition, auxin plays crucial roles in gravitropism (roots growing in a downward direction), patterning and development. These behaviors all rely on Polar Auxin Transport (PAT), which is an ability of the plant to transport auxin across its various tissues to the site where it stimulates cell division, expansion and differentiation. For this process, the plant has specific membrane proteins that belong to the PIN family. The colored arrows on the illustration represent transport by various family members. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656606322750-CDILBRWT4G73FGP4DBKE/auxins.png Home - Auxin transport by PIN8 in plants - Auxins represent a class of small molecules The presence of a hormone that controls plant growth has been a topic of study for 250 years. In 1880, Darwin postulated that something was transmitted to one side of the plant, causing it to bend toward the light. In the 1920’s and 1930’s, Frits Went identified indole acetic acid (IAA), which is indeed the prevalent auxin in plants and is synthesized from tryptophan. Transport of IAA by PIN transporters was established in the 1990’s. In addition to IAA, there exist a variety of other natural auxins as well as synthetic agonists and antagonists used both for experimental work and as herbicides. In particular, naphthylphthalamic acid (NPA) was identified as a potent inhibitor of growth in the 1950’s, although its specific association with PIN transporters has only recently been established. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656608085035-INOT29K7R4NYNH3P3Z1R/pin-family.png Home - Auxin transport by PIN8 in plants - PIN proteins responsible for auxin transport Mutation of PIN1 in Arabadopsis perturbs formation of the central stalk and flowering (inflorescence). PIN2 controls downward grown of roots. The family includes eight different homologs with canonical (1, 2, 3, 4, 7) and non-cononical (5, 6, 8) members distinguished by the length of an intracellular loop. This loop is implicated in phospho-regulation of activity, with canonical members requiring phosphatase activity for activation in Xenopus oocyte assays. In collaboration with the laboratory of Bjørn Pedersen at Aarhus University and the laboratory of Ulrich Hammes at the Technical University of Munich, we are studying the non-canonical PIN8, which has a short intracellular loop and appears to be constitutively active. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656609458661-CAR9SYSJU0SYW8P0GY4M/pin_cryoem_structure.png Home - Auxin transport by PIN8 in plants - Cryo-EM structure of PIN8 Cryo-electron microscopy was used to solve structures of PIN8, thus revealing a dimeric complex. Each protein comprised a pseudo-repeat of five transmembrane helices which assembles into a scaffold domain and a transport domain. An interrupted helix (4a & 4b) produces a cross-over motif at the interface between these two domains and lies at the center of the substrate binding pocket. The structure of the apo state represents an outward facing conformation in which the empty substrate binding site faces the outside of the cell. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1656609709068-UR9SU6XGBQH18QP0S93S/pin_elevator_mech.png Home - Auxin transport by PIN8 in plants - PIN8 operates via an elevator mechanism Additional cryo-EM structures were solved in the presence of the canonical auxin, IAA, as well as the inhibitor and herbicide, NPA. The latter was in an inward-facing conformation revealing domain movements associated with transport. In particular, movement of the transport domain relative to the scaffold domain not only alternates accessibility of the binding pocket, but also displaces the substrate relative to the membrane plane. Such movements have been described as an elevator mechanism and have been seen in transporters of a variety of other substrates: e.g., bile acids, and exchange of bicarbonate/sodium and sodium/protons. https://www.stokeslabnyu.org/home/kdpfabc monthly 0.5 2022-06-16 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655480965797-OELAZA8LAP7D4P4PFP00/K-transport-systems.png Home - Potassium transport by KdpFABC in bacteria - Potassium transport systems in bacteria Organisms from all kingdoms of life maintain elevated levels of potassium in the cytoplasm of their cells. The resulting K+ gradient is used to maintain membrane potential, osmotic pressure and pH. The gradient is used as an energy source for secondary transport systems and is coupled to cell growth and division. In bacteria there are multiple systems for maintaining potassium homeostasis. Under normal growth conditions, bacteria rely on constitutively expressed uptake systems (Trk and Kup), but when K+ levels fall into the micromolar range, the kdp operon governs expression of the KdpFABC membrane complex. This complex serves as an ATP-dependent transporter that actively drives K+ into the cell. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655481755593-WSYW6QT91L3WL6RKJIBN/kdp-topology.png Home - Potassium transport by KdpFABC in bacteria - KdpFABC is a complex comprising a pump and a channel The KdpFABC complex consists of four subunits that work together to couple ATP hydrolysis to the uphill transport of K+ across the membrane. KdpA is a channel-like subunit that belongs to the Superfamily of K+ Transporters. KdpB is a pump-like subunit that belongs to the P-type ATPase superfamily. KdpC and KdpF are single-pass membrane proteins with no obvious homologs outside of the Kdp system. Like the beta subunits of other P-type ATPases, they most likely serve as structural chaperones to stabilize the complex. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655483925406-WF10DNZH167QTJIQVKMG/kdp-overview.png Home - Potassium transport by KdpFABC in bacteria - Architecture of KdpFABC Our initial structure of the KdpFABC complex was solved by X-ray crystallography. It shows expected architectures for a K+ channel (KdpA, green) and a P-type pump (KdpB), which is characterized by a group of 7 transmembrane helices (brown) and 3 cytoplasmic domains (A-domain in yellow, N-domain in red, P-domain in blue). Surprisingly, this structure revealed a phosphorylated serine residue on the A-domain mediating a salt bridge with the N-domain. Subsequent studies showed that serine phosphorylation represents a regulatory mechanism for inhibiting the pump when K+ is restored to the growth medium such that pumping by KdpFABC is no longer required. https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655487772458-I54AAI0YVXQ0LYEBN7I7/kdp_tunnel.png Home - Potassium transport by KdpFABC in bacteria https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655482420857-R5Z9V20PHEKCH5VZEJ93/Ptype_family_tree.png Home - Potassium transport by KdpFABC in bacteria https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655482646785-SMAVXDL1V2KDJTV5GTP5/SKT_evolution.png Home - Potassium transport by KdpFABC in bacteria https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655486384478-CXGSVJOU98ZBTTSGK2SB/Fig1b_Apo.png Home - Potassium transport by KdpFABC in bacteria https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655486538058-DLIRZAPHY3S2VMT2CIC7/Fig1b_ACP.png Home - Potassium transport by KdpFABC in bacteria https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655486740057-Y280EKH5OBK6F7R0NT9G/Fig1b_Mg.png Home - Potassium transport by KdpFABC in bacteria https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1655486866404-STUVLWCW99RLVI36P511/Fig1b_MgF.png Home - Potassium transport by KdpFABC in bacteria https://www.stokeslabnyu.org/home/tafazzin monthly 0.5 2022-06-16 https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/f593b08b-0cd2-4a3e-af81-03e3de7fae10/TZ-biosynthesis_simple.png https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/1365c290-e9e1-4b88-8bcf-1dadc98c6bc8/mitofig2a.png https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/5ade8782-9329-42bc-bde7-9ee7a39b7283/mitofig3a01.png https://images.squarespace-cdn.com/content/v1/62aa3fa8d7067d186dff4c4f/528cb80f-3429-4916-a56d-7e906d94678b/GPAT_structures.png