Robust Sequence Determinants of α‑Synuclein Toxicity in Yeast Implicate Membrane Binding

Protein
conformations are shaped by cellular environments, but how environmental
changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to
the challenge of probing protein structures in living cells. Here,
we use deep mutational scanning to investigate how a toxic conformation
of α-synuclein, a dynamic protein linked to Parkinson’s
disease, responds to perturbations of cellular proteostasis. In the
context of a course for graduate students in the UCSF Integrative
Program in Quantitative Biology, we screened a comprehensive library
of α-synuclein missense mutants in yeast cells treated with
a variety of small molecules that perturb cellular processes linked
to α-synuclein biology and pathobiology. We found that the conformation
of α-synuclein previously shown to drive yeast toxicityan
extended, membrane-bound helixis largely unaffected by these
chemical perturbations, underscoring the importance of this conformational
state as a driver of cellular toxicity. On the other hand, the chemical
perturbations have a significant effect on the ability of mutations
to suppress α-synuclein toxicity. Moreover, we find that sequence
determinants of α-synuclein toxicity are well described by a
simple structural model of the membrane-bound helix. This model predicts
that α-synuclein penetrates the membrane to constant depth across
its length but that membrane affinity decreases toward the C terminus,
which is consistent with orthogonal biophysical measurements. Finally,
we discuss how parallelized chemical genetics experiments can provide
a robust framework for inquiry-based graduate coursework.