The protein synthesis inhibitor blasticidin s enters mammalian cells via leucine-rich repeat-containing protein 8D

Abstract

Leucine-rich repeat-containing 8 (LRRC8) proteins have been identified as putative receptors involved in lymphocyte development and adipocyte differentiation. They remain poorly characterized, and no specific function has been assigned to them. There is no consensus on how this family of proteins might function because homology searches suggest that members of the LRRC8 family act not as plasma membrane receptors, but rather as channels that mediate cell-cell signaling. Here we provide experimental evidence that supports a role for LRRC8s in the transport of small molecules. We show that LRRC8D is a mammalian protein required for the import of the antibiotic blasticidin S. We characterize localization and topology of LRRC8A and LRRC8D and demonstrate that LRRC8D interacts with LRRC8A, LRRC8B, and LRRC8C. Given the suggested involvement in solute transport, our results support a model in which LRRC8s form one or more complexes that may mediate cell-cell communication by transporting small solutes.

Keywords: Antibiotics; Blasticidin S; Drug Transport; Gene Knockout; LRRC8D; Mass Spectrometry (MS); Membrane Protein; Metabolomics; Protein-Protein Interaction; Somatic Cell Genetics.

FIGURE 1.

LRRC8D-deficient cells are resistant to blasticidin S. A, structure of BlaS. B, genomic locus of LRRC8D and the location of two gene trap insertion sites. White boxes denote the 5′- and 3′-untranslated regions, and the black box denotes the coding sequence, all of which is within exon 3. Arrowheads indicate the binding sites of primers used for RT-PCR analysis. C, RT-PCR analysis of WT KBM7 cells, WT NF-κB-BSR reporter cells, and two clonally derived cell lines containing gene trap insertions in LRRC8D as indicated in B. D, WT KBM7 cells, WT NF-κB reporter cells, LRRC8D^GT1 cells, and LRRC8D^GT2 cells treated with varying concentrations of BlaS. Cell viability was determined after 24 h of treatment using the CellTiter Glo assay, and results are plotted as percentage viability of treated cells compared with untreated cells. Results are mean ± S.E. (error bars) of triplicates and are representative of three independent experiments.

FIGURE 2.

BlaS internalization is defective in LRRC8D-deficient cells. WT reporter cells, LRRC8D^GT1 cells, and LRRC8D^GT2 cells were treated with varying concentrations of BlaS for 3 h. Extracts of polar metabolites from the cells were analyzed for the presence of BlaS using LC/MS. Results are mean ± S.D. (error bars) of triplicates and are representative of three independent experiments. n.d., not detected. Statistical analysis was performed using the Welch test followed by a Bonferroni correction (*, p < 0.0001).

FIGURE 3.

Localization of LRRC8A and LRRC8D. HeLa cells stably expressing human LRRC8A or LRRC8D C-terminally tagged with a HA epitope tag were stained for HA, the ER, and actin and examined by confocal microscopy. Images are representative of three independent experiments. Scale bars, 7 μm.

FIGURE 4.

Topology of LRRC8A and LRRC8D. A, schematic depicts the topology of KEL-HA and GYPA-HA at the plasma membrane. Also depicted are the predicted topology of LRRC8A-HA and LRRC8D-HA at the plasma membrane based upon results in B. B, HeLa cells stably expressing either KEL-HA, GYPA-HA, LRRC8A-HA, LRRC8D-HA, or empty vector were incubated with Alexa Fluor 488-conjugated anti-HA in the absence or presence of saponin. C, schematic depicts the experimental set-up in D, the topology of HA-SEL1L and AUP1-GFP in the ER, and the predicted topology of LRRC8D-HA based upon results in D. D, HA-SEL1L, AUP1-GFP, and LRRC8D-HA were transiently expressed in 293T cells. A microsome-containing fraction from these cells was exposed to proteinase K in the absence or presence of Triton X-100 followed by immunoblot analysis. Results are representative of two (B) and three (D) independent experiments.

FIGURE 5.

LRRC8D interacts with LRRC8A, B, and C. 293T cells were transiently transfected with murine LRRC8A-Myc, LRRC8B-V5, LRRC8C-FLAG, and LRRC8D-HA. + indicates that constructs were coexpressed in the same cells. / indicates that constructs were independently expressed in separate cells and the lysates were combined post-lysis as a control. Blots are representative of two independent experiments.