Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1

A sustained ocean observing system in the Indian Ocean for climate related scientific knowledge and societal needs

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hermes, J. C., Masumoto, Y., Beal, L. M., Roxy, M. K., Vialard, J., Andres, M., Annamalai, H., Behera, S., D'Adamo, N., Doi, T., Peng, M., Han, W., Hardman-Mountford, N., Hendon, H., Hood, R., Kido, S., Lee, C., Lees, T., Lengaigne, M., Li, J., Lumpkin, R., Navaneeth, K. N., Milligan, B., McPhaden, M. J., Ravichandran, M., Shinoda, T., Singh, A., Sloyan, B., Strutton, P. G., Subramanian, A. C., Thurston, S., Tozuka, T., Ummenhofer, C. C., Unnikrishnan, A. S., Venkatesan, R., Wang, D., Wiggert, J., Yu, L., & Yu, W. (2019). A sustained ocean observing system in the Indian Ocean for climate related scientific knowledge and societal needs. Frontiers in Marine Science, 6, (2019): 355, doi: 10.3389/fmars.2019.00355.The Indian Ocean is warming faster than any of the global oceans and its climate is uniquely driven by the presence of a landmass at low latitudes, which causes monsoonal winds and reversing currents. The food, water, and energy security in the Indian Ocean rim countries and islands are intrinsically tied to its climate, with marine environmental goods and services, as well as trade within the basin, underpinning their economies. Hence, there are a range of societal needs for Indian Ocean observation arising from the influence of regional phenomena and climate change on, for instance, marine ecosystems, monsoon rains, and sea-level. The Indian Ocean Observing System (IndOOS), is a sustained observing system that monitors basin-scale ocean-atmosphere conditions, while providing flexibility in terms of emerging technologies and scientificand societal needs, and a framework for more regional and coastal monitoring. This paper reviews the societal and scientific motivations, current status, and future directions of IndOOS, while also discussing the need for enhanced coastal, shelf, and regional observations. The challenges of sustainability and implementation are also addressed, including capacity building, best practices, and integration of resources. The utility of IndOOS ultimately depends on the identification of, and engagement with, end-users and decision-makers and on the practical accessibility and transparency of data for a range of products and for decision-making processes. Therefore we highlight current progress, issues and challenges related to end user engagement with IndOOS, as well as the needs of the data assimilation and modeling communities. Knowledge of the status of the Indian Ocean climate and ecosystems and predictability of its future, depends on a wide range of socio-economic and environmental data, a significant part of which is provided by IndOOS.This work was supported by the PMEL contribution no. 4934

Trichosporon loubieri Infection in a Patient with Adult Polycystic Kidney Disease

A 45-year-old man from Nepal with a 13-year history of polycystic kidney disease was diagnosed as suffering from chronic renal failure with end-stage renal disease. After receiving empirical antituberculosis treatment, he was treated with broad-spectrum antibiotics. A left nephrectomy was performed, and after 4 months, he received a kidney transplant. The left kidney was grossly enlarged, with multiple cystic spaces filled with blackish material. Histologic examination of the excised left kidney tissue stained with hematoxylin and eosin and Gomori's methenamine silver stains showed numerous hyaline, septate, fungal hyphae of various lengths, many broken into rectangular arthroconidia in the cystic spaces. Culture of the kidney tissue yielded white, glabrous, yeast-like colonies. Based on its micromorphology, growth at 42°C, and ribosomal DNA (rDNA) sequence analysis, and also sequence analysis of the internal-transcribed-spacer and D1/D2 rDNA regions, the yeast was identified as Trichosporon loubieri. Postsurgically, the patient was treated with amphotericin B and oral itraconazole, followed by maintenance therapy with fluconazole. He remained afebrile and asymptomatic. At the final follow-up, all parameters were found normal and the patient was doing well, with normal renal function reports. This paper presents the first known case of human infection caused by T. loubieri

Biochemical Characterization of UDP-<i>N</i>-acetylmuramoyl-L-alanyl-D-glutamate: <i>meso</i>-2,6-diaminopimelate ligase (MurE) from <i>Verrucomicrobium spinosum</i> DSM 4136^T

<div><p><i>Verrucomicrobium spinosum</i> is a Gram-negative bacterium that is related to bacteria from the genus <i>Chlamydia</i>. The bacterium is pathogenic towards <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i>, using a type III secretion system to facilitate pathogenicity. <i>V. spinosum</i> employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the <i>V. spinosum</i> genome provides evidence that the bacterium should be able to synthesize peptidoglycan <i>de novo</i>, since all of the necessary genes are present. The enzyme UDP-<i>N</i>-acetylmuramoyl-l-alanyl-d-glutamate: <i>meso</i>-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.15) catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The <i>murE</i> ortholog from <i>V. spinosum</i> (<i>murE</i>_Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:<i>meso</i>-2,6-diaminopimelate ligase activity <i>in vivo</i> using functional complementation. <i>In vitro</i> analysis using the purified recombinant enzyme demonstrated that MurE_Vs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. <i>meso</i>-Diaminopimelate was the preferred substrate with a <i>K</i>_m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurE_Vs. Our kinetic analysis and structural model of MurE_Vs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that <i>V. spinosum</i> incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a <i>V. spinosum</i> culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.</p></div

The monomer unit of the peptidoglycan structure.

<p>The disaccharide moiety is composed of the amino sugars <i>N</i>-acetylglucosamine (GlcNAc) and <i>N</i>-acetylmuramic (MurNAc) linked via a β-1,4 glycosidic bond. The amino acid at position 3 of the stem peptide is <i>meso</i>-diaminopimelic acid (R =  COOH) in most Gram-negative bacteria and l-lysine (R = H) in most Gram-positive bacteria.</p

Homolgy model of MurE_Vs.

<p>(a) The homology model of MurE_Vs highlighting domains A (grey), B (violet) and C (pink). (b) Shows the structure model of MurE_Vs bound to UDP-MurNAc-tripeptide (UMT) product (yellow). (c) Active site residue hypothesized to bind to UMT product is shown in red. The structure has been rotated 90° on the right panel for the better viewing of the binding pocket. (d) Cross eye stereo view showing the interaction between amino acid residues of the binding site and UMT product.</p

Expression and purification of recombinant MurE_Vs using His-tag affinity chromatography.

<p>Lane (1) protein makers (kDa); Lane (2) 10 µg of soluble protein from uninduced cells; Lane (3) 10 µg of soluble protein from induced cells; Lane (4) 1 µg of purified recombinant MurE_Vs. The proteins were resolved on 10% (w/v) acrylamide gel and were stained using Coomassie blue.</p

List of genes involved in PG metabolism of <i>V. spinosum</i> DSM 4136^T.

<p>The annotated gene product names are from NCBI (<a href="http://www.ncbi.nlm.nih.gov/protein/" target="_blank">www.ncbi.nlm.nih.gov/protein/</a>) queried of February 28, 2013. The pencillin-binding proteins (PBP) class designations are denoted by activity based on <b><u>p</u></b>rotein <b><u>fam</u></b>ily (pfam) domains. Class A and class B PBPs are high-molecular mass PBPs while class C PBPs are low-molecular mass PBPs. Class A PBPs are predicted to have both transglycosylase and transpeptidase activities; class B PBPs are predicted to have only transpeptidase activity; class C PBPs are predicted to have d,d-carboxypeptidase activity.</p

Multiple amino acid sequence alignment of five representative sequences of MurE.

<p>The residues that are predicted to be involved in binding in the active site are marked with a star below the sequence. The sequence identity score against MurE from <i>V. spinosum</i> was: <i>C. trachomatis,</i> 37%; <i>E. coli,</i> 35%; <i>P. carotovorum</i>, 36%; and <i>M. tuberculosis</i>. The multiple amino acid sequence alignment figure was generated using the ESPript 2.2 server (<a href="http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi" target="_blank">http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi</a>).</p

Analysis of crude and purified PG from <i>V. spinosum</i> DSM 4136^T.

a<p>Crude and purified PG designate the macromolecule before and after, respectively, treatment with pancreatin, pronase and trypsin (see Materials and Methods).</p