Mutations in <i>dapA</i> restore growth to <i>ridA</i> mutants in the presence of serine.

<p>Growth was monitored over time as optical density at 650 nm. Strains were grown at 37°C in minimal glucose medium with no additions (closed symbols) or 5 mM serine (open symbols). Shown are strains <i>ridA</i> (DM3480), squares; <i>ridA dapA356</i> (DM11637), triangles; and <i>ridA dapA360</i> (DM11640), circles. Curves displayed were representative of 3 biological replicates.</p

Pathway for synthesis of aspartate-derived amino acids.

<p>Aspartate is a precursor to lysine, methionine, threonine, and isoleucine, as depicted here. Aspartate 4-semialdehyde (ASA) is a branchpoint metabolite controlled by the activities of DapA, ThrA, and MetL.</p

IlvA variants have reduced activity.

*<p>Threonine dehydratase (IlvA) activity measured in crude extracts from DM3480 (<i>ridA</i>), DM7610 (<i>ridA ilvA3210</i>) and DM7608 (<i>ridA ilvA3211</i>) and reported as ΔA_{540 nm}/min/mg protein.</p>†<p>Growth rate (in h⁻¹) (µ =  ln(X/X0)/T where X =  optical density at 650 nm and T =  time in hours during logarithmic growth) for strains DM10332 (WT), DM10331 (<i>ilvA3210</i>), and DM11558 (<i>ilvA3211</i>) determined from growth in minimal medium with glucose (Glc) and glucose with isoleucine (Glc Ile).</p>‡<p>Below Detection.</p

The ThrA_G403D variant is insensitive to feedback inhibition by threonine and serine.

*<p>Homoserine dehydrogenase activity was measured in crude extracts from isogenic strains DM11877 (<i>ridA thrA1371</i>) and DM11878 (<i>ridA</i>) by following reduction of NADP+ and was reported as ΔA_{420 nm}/min/µg protein.</p

Suppressing DapA variants have decreased specific activities.

*<p>A <i>ridA</i> strain carrying any of the listed alleles is able to grow in the presence of serine.</p>†<p>Specific activity of DapA in µmol NADPH oxidized/sec/mg of purified protein.</p>‡<p>N.D.  =  not determined.</p

Bacterial strains.

*<p>MudJ refers to Mud1734 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043082#pone.0043082-Castilho1" target="_blank">[37]</a>. Tn<i>10</i>d(Tc) refers to the transposition-defective mini-Tn<i>10</i>(Tn<i>10</i>Δ16Δ17 <i>tet^R</i>) construct <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043082#pone.0043082-Way1" target="_blank">[38]</a>.</p

Suppressor mutations increase growth in <i>purF ridA</i> strain background.

<p>Strains were grown at 37°C in minimal glucose medium with adenine (open symbols) or further supplemented with 0.3 mM threonine (closed symbols). Growth was monitored over time as optical density at 650 nm. Shown are strains <i>purF ridA</i> (DM3871), triangles; <i>purF ridA thrA1371</i> (DM6309), diamonds; and <i>purF ridA dapA356</i> (DM11412), circles. Error bars represent standard deviations of three biological replicates.</p

Adaptor protein complexes AP-1 and AP-3 are required by the HHV-7 Immunoevasin U21 for rerouting of class I MHC molecules to the lysosomal compartment.

The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes

Boron Isotopes in Fresh Surface Waters in a Temperate Coastal Setting

The results from a four-year study of a freshwater pond on Long Island, NY, USA, do not point to a single source of boron (and by proxy other elements including nutrients) in this system. However, boron data from samples associated with this pond can be explained by mixing between average precipitation (weighted average δ11B = 22.7) in the area and the local sources of boron, both natural and anthropogenic. This multiyear study provided the opportunity to see both yearly and seasonal differences. One algae sample from the pond showed significant fractionation and enrichment in light boron relative to the water and suggests algae may act as a boron sink. This type of biological fractionation could explain an observed down-gradient trend to heavier boron isotope values in pond water, which corresponds to the slight reduction in boron concentration seen in 2021. However, the trend was subdued in the following year, likely due to differences in the water flow rates and/or rate of algal growth. An opposite trend was seen with depth in the water, where δ11B showed a positive correlation to boron concentration, which increased with depth from the surface of the pond. This gradient may be explained by the stratification of the pond with a heavy source concentrating in the bottom waters. The bottom water composition was consistent with goose feces (δ11B = 25.8) or the addition of chemicals from the application of rock salt to local roads in winter. Surprisingly, boron from seawater (average δ11B = 39.8) did not appear to have a direct impact on Setauket Pond, other than its influence on precipitation, providing heavy δ11B and very low boron concentrations

MCMV gp48-mediated trafficking of class I MHC molecules is affected by depletion of AP-1μ or AP-3μ subunits.

<p>Immunofluorescence analysis of class I MHC molecules in cells expressing gp48HA, − (a) and + (b) AP-1μ siRNA, and - (d) and + (e) AP-3μ siRNA, as indicated. Arrows in panels b and e point to the plasma membrane. Scale bar = 10 µm. c and f) Immunoblot analysis of AP-1μ or AP-3μ from lysates of U373-gp48HA cells −/+ treatment with AP-1μ (c) or AP-3μ (f) siRNA, as indicated. The Ponceau S stained nitrocellulose is shown beneath the immunoblots as a loading control.</p