Accelerating and de-risking CMC development with transposon-derived manufacturing cell lines.

The development of highly productive, genetically stable manufacturing cell lines is on the critical path to IND filing for protein-based biologic drugs. Here, we describe the Leap-In Transposase® platform, a novel transposon-based mammalian (e.g., Chinese hamster ovary) cell line development system that produces high-titer stable pools with productivity and product quality attributes that are highly comparable to clones that are subsequently derived therefrom. The productivity distributions of clones are strongly biased toward high producers, and genetic and expression stability is consistently high. By avoiding the poor integration rates, concatemer formation, detrimental transgene recombination, low average expression level, unpredictable product quality, and inconsistent genetic stability characteristic of nonhomologous recombination methods, Leap-In provides several opportunities to de-risk programs early and reduce timelines and resources.

Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3.

Maturation of organelles in the endolysosomal pathway requires exchange of the early endosomal GTPase Rab5/Vps21 for the late endosomal Rab7/Ypt7. The Rab exchange depends on the guanine nucleotide exchange factor activity of the Mon1-Ccz1 heterodimer for Ypt7. Here we investigate vacuole binding and recycling of Mon1-Ccz1. We find that Mon1-Ccz1 is absent on vacuoles lacking the phosphatidic acid phosphatase Pah1, which also lack Ypt7, the phosphatidylinositol 3-kinase Vps34, and the lipid phosphatidylinositol 3-phosphate (PI3P). Interaction of Mon1-Ccz1 with wild-type vacuoles requires PI3P, as shown in competition experiments. We also find that Mon1 is released from vacuoles during the fusion reaction and its release requires its phosphorylation by the type 1 casein kinase Yck3. In contrast, Mon1 is retained on vacuoles lacking Yck3 or when Mon1 phosphorylation sites are mutated. Phosphorylation and release of Mon1 is restored with addition of recombinant Yck3. Together the results show that Mon1 is recruited to endosomes and vacuoles by PI3P and, likely after activating Ypt7, is phosphorylated and released from vacuoles for recycling.

The yeast ATP-binding cassette (ABC) transporter Ycf1p enhances the recruitment of the soluble SNARE Vam7p to vacuoles for efficient membrane fusion.

The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd(2+) transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p(K669M)) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.

The lipid composition and physical properties of the yeast vacuole affect the hemifusion-fusion transition.

Yeast vacuole fusion requires the formation of SNARE bundles between membranes. Although the function of vacuolar SNAREs is controlled in part by regulatory lipids, the exact role of the membrane in regulating fusion remains unclear. Because SNAREs are membrane-anchored and transmit the force required for fusion to the bilayer, we hypothesized that the lipid composition and curvature of the membrane aid in controlling fusion. Here, we examined the effect of altering membrane fluidity and curvature on the functionality of fusion-incompetent SNARE mutants that are thought to generate insufficient force to trigger the hemifusion-fusion transition. The hemifusion-fusion transition was inhibited by disrupting the 3Q:1R stoichiometry of SNARE bundles with the mutant SNARE Vam7p(Q283R) . Similarly, replacing the transmembrane domain of the syntaxin homolog Vam3p with a lipid anchor allowed hemifusion, but not content mixing. Hemifusion-stalled reactions containing either of the SNARE mutants were stimulated to fuse with chlorpromazine, an amphipathic molecule that alters membrane fluidity and curvature. The activity of mutant SNAREs was also rescued by the overexpression of SNAREs, thus multiplying the force transferred to the membrane. Thus, we conclude that either increasing membrane fluidity, or multiplying SNARE-generated energy restored the fusogenicity of mutant SNAREs that are stalled at hemifusion. We also found that regulatory lipids differentially modulated the complex formation of wild-type SNAREs. Together, these data indicate that the physical properties and the lipid composition of the membrane affect the function of SNAREs in promoting the hemifusion-fusion transition.

SNAREs, HOPS and regulatory lipids control the dynamics of vacuolar actin during homotypic fusion in S. cerevisiae.

Homotypic vacuole fusion requires SNAREs, the Rab Ypt7p, the tethering complex HOPS, regulatory lipids and actin. In Saccharomyces cerevisiae, actin functions at two stages of vacuole fusion. Pre-existing actin filaments are depolymerized to allow docking and assembly of the vertex ring (a microdomain enriched in proteins and lipids that mediate fusion). Actin is then polymerized late in the pathway to aid fusion. Here, we report that the fusion machinery regulates the accumulation of actin at the vertex ring. Using Cy3-labeled yeast actin to track its dynamics, we found that its vertex enrichment was abolished when actin monomers were stabilized by latrunculin-B, independent of the extent of incorporation. By contrast, stabilization of filamentous actin with jasplakinolide markedly augmented actin vertex enrichment. Importantly, agents that inhibit SNAREs, Ypt7p and HOPS inhibited the vertex enrichment of actin, demonstrating that the cytoskeleton and the fusion machinery are interdependently regulated. Actin mobilization was also inhibited by ligating ergosterol and PtdIns(3)P, whereas the ligation or modification of PtdIns(4,5)P(2) augmented the vertex enrichment of actin. The proteins and lipids that regulated actin mobilization to the vertex did not affect the total incorporation of Cy3-actin, indicating that actin mobilization and polymerization activities can be dissociated during membrane fusion.

Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion.

Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1p(D398E) had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion.