A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents

The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the alkylphosphocholines exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of Saccharomyces cerevisiae titratable-essential gene and haploid non-essential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). 58 strains out of ∼5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum (ER) and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents

The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the alkylphosphocholines exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of Saccharomyces cerevisiae titratable-essential gene and haploid non-essential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). 58 strains out of ~5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum (ER) and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.

Cross-bridges mediate anterograde and retrograde vesicle transport along microtubules in squid axoplasm.

To assay the detailed structural relationship between axonally transported vesicles and their substrate microtubules, vesicle transport was focally cold blocked in axoplasm that was extruded from the squid giant axon. A brief localized cold block concentrated anterogradely and retrogradely transported vesicles selectively on either the proximal or or distal side of the block. Normal movement of the concentrated vesicles was reactivated by rewarming the cold-blocked axoplasm. At the periphery of the axoplasm, moving vesicles were located on individual microtubules that had become separated from the other cytomatrix components. The presence of moving vesicles on isolated microtubules permitted the identification of the structural components required for vesicle transport along microtubules. The results show that 16-18-nm cross-bridges connect both anterogradely and retrogradely moving vesicles to their substrate microtubules. These observations demonstrate that cross-bridges are fundamental are fundamental components of vesicle transport along axonal microtubules. Thus, vesicle transport can now be included among those cell motile systems such as muscle and axonemes that are based on a cross-bridge-mediated mechanism.