The interactome of multimeric recombinant proteins in yeast cells with modified ER morphology

Background.

Proteins destined for secretion in eukaryotes have to pass through the secretory pathway, an endomembrane system, where they undergo folding and post-translational modifications (PTM). Recombinant proteins interact with many host proteins on their way through the secretory pathway, e.g. chaperones, enzymes and receptors, which aid the correct assembly, modifications and transport.
We have previously established methods to study the interactome (i.e., interacting host cell proteome) of heterologous recombinant proteins in the yeast Komagataella phaffii (syn Pichia pastoris) (Pfeffer, 2012), and successfully used the generated knowledge to determine and overcome cellular bottlenecks in protein secretion (Zahrl, 2022).

The initial destination for secretory proteins is the endoplasmic reticulum (ER), a peripheral network of tubules and membrane sheets (Perkins, 2021). The reticulon (RTN) and DP1/Yop1p protein families are responsible for tubule formation and regulate the ratio of sheets to tubules, while the dynamin-like GTPase atlastin/Sey1p is required for homotypic ER fusion and three-way junctions of the ER network, together with other auxiliary proteins. The ER is a very crowded and spatially restricted organelle, where proteins can also interact with wrong partners and aggregate. This causes ER stress. Increasing ER size through lipid-engineering in yeast lead to increased secretion of recombinant proteins (de Ruijter, 2016) and reduced ER stress independently of chaperone levels (Schuck, 2009). The impact of ER morphology in these processes remains so far undetermined.

Aims & Hypotheses.

Systematically study the impact of ER morphology changes (i.e., sheets versus tubules) on the fate of (recombinant) secretory proteins. It is hypothesized that expanding the ER size (luminal volume) and altering the ER morphology will influence (1) the interactome of recombinant proteins with host cell proteins and (2) the ability to secrete complex recombinant proteins in K. phaffii, as well as (3) the ER stress exerted upon their expression.

Methods.

Generation of K. phaffii ER morphology mutants by overexpressing or disrupting the respective genes (selected based on literature research), using state-of-the-art cloning methods (such as CRISPR/Cas9, Golden Gate Assembly). Verification of morphological changes by microscopic imaging, esp. electron microscopy or fluorescence microscopy.
The secretion efficiency and interactome of two differently complex recombinant proteins (dimeric Fab antibody Fab fragments, scFv/vHH, or Fc fusion protein) will be tested in selected ER morphology mutants.
K. phaffii strains will be cultivated in established small scale screenings and bioreactor cultivations (Zahrl, 2022), the recombinant proteins in the supernatant will be quantified by capillary electrophoresis or ELISA, while intracellularly retained proteins will be determined by Western Blot or immunostaining.
The interactome will be determined from cell lysates by co-immunoprecipitation (co-IP) followed by proteomics (Pfeffer, 2012), with LC-MS/MS analyses done by BOKU core facility. Live cell imaging of fluorescently tagged organelle markers and recombinant proteins will be used as complementary approach to study the nature and kinetics of interaction partners.

REFERENCES
1. Pfeffer, M., M. Maurer, J. Stadlmann, J. Grass, M. Delic, F. Altmann and D. Mattanovich (2012) Intracellular interactome of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation. Appl Microbiol Biotechnol 93, 2503-2512. doi: 10.1007/s00253-012-3933-3
2. Zahrl, R. J., R. Prielhofer, O. Ata, K. Baumann, D. Mattanovich and B. Gasser (2022) Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion. Metab Eng 74, 36- 48. doi: 10.1016/j.ymben.2022.08.010
3. Perkins, H. T. and V. Allan (2021) Intertwined and Finely Balanced: Endoplasmic Reticulum Morphology, Dynamics, Function, and Diseases. Cells 10. doi: 10.3390/cells10092341
4. de Ruijter, J. C., E. V. Koskela and A. D. Frey (2016) Enhancing antibody folding and secretion by tailoring the S. cerevisiae endoplasmic reticulum. Microb Cell Fact 15, 87. doi: 10.1186/s12934-016-0488-5
5. Schuck, S., W. A. Prinz, K. S. Thorn, C. Voss and P. Walter (2009) Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response. J Cell Biol 187, 525-536. doi: 10.1083/jcb.200907074