A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key mediator in the response to tunicamycin

Abstract

Tunicamycin (TM) inhibits eukaryotic asparagine-linked glycosylation, protein palmitoylation, ganglioside production, proteoglycan synthesis, 3-hydroxy-3-methylglutaryl coenzyme-A reductase activity, and cell wall biosynthesis in bacteria. Treatment of cells with TM elicits endoplasmic reticulum stress and activates the unfolded protein response. Although widely used in laboratory settings for many years, it is unknown how TM enters cells. Here, we identify in an unbiased genetic screen a transporter of the major facilitator superfamily, major facilitator domain containing 2A (MFSD2A), as a critical mediator of TM toxicity. Cells without MFSD2A are TMresistant, whereas MFSD2A-overexpressing cells are hypersensitive. Hypersensitivity is associated with increased cellular TM uptake concomitant with an enhanced endoplasmic reticulum stress response. Furthermore, MFSD2A mutant analysis reveals an important function of the C terminus for correct intracellular localization and protein stability, and it identifies transmembrane helical amino acid residues essential for mediating TM sensitivity. Overall, our data uncover a critical role for MFSD2A by acting as a putative TM transporter at the plasma membrane.

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

“…We performed a loss-of-function genetic screen in human haploid cells to identify mutant cell lines resistant to Chlamydia (13)(14)(15)(16). This system avoids the potential off-target effects and incomplete knockdowns of an siRNA screen.…”

Knocking out the door to tunicamycin entry

“…11). Reiling et al (8) show that MFSD2A is localized to the plasma membrane and that this localization is required for its function, consistent with a role in transport (Fig. 1A).…”

“…Despite its widespread use in the laboratory, the mechanism by which tunicamycin enters cells has remained a mystery. In PNAS, Reiling et al (8) investigate the requirements for tunicamycin sensitivity in mammalian cells and identify MFSD2A as a putative plasma membrane transporter.…”

“…To identify factors required for tunicamycin-induced death, Reiling et al (8) use an insertional mutagenesis strategy in the near-haploid human cell line KBM7. This remarkable technique, first reported by Carette et al (9,10), can yield complete gene disruptions at targeted loci, whose site of insertion can then be identified by sequencing.…”

Haploid animal cells

“…Both cell systems have distinct strengths and weaknesses. For example, screening in human tumor cells can identify genes in a clinically relevant genome (Birsoy et al, 2013;Carette et al, 2009;Carette et al, 2011;Guimaraes et al, 2011;Reiling et al, 2011;Rosmarin et al, 2012), but tumor-specific genetic aberrations and oncogenic signals could interfere with the particular aim of a genetic screen. The stable haploid karyotype of human tumor cells and ease of culture are additional technical advantages over haploid mouse ESCs.…”