A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris

Nuclear localization sequences (NLSs) are required for the import of proteins in the nucleus of eukaryotes. However many proteins from bacteria or bacteriophages are used for basic studies in molecular biology, to generate synthetic genetic circuits or for genome editing applications. Prokaryotic recombinases, CRISPR-associated proteins such as Cas9 or bacterial and viral polymerases require efficient NLSs to function in eukaryotes. The yeast Pichia pastoris is a widely used expression platform for heterologous protein production, but molecular tools such as NLSs are limited. Here we have characterized a set of 10 NLSs for P. pastoris, including the first endogenous NLSs (derived from P. pastoris proteins) and commonly used heterologous NLSs. The NLSs were evaluated by fusing them in N- and C-terminal position to an enhanced green fluorescent protein showing pronounced differences in fluorescence levels and nuclear targeting. Thereby we provide a set of different NLSs that can be applied to optimize the nuclear import of heterologous proteins in P. pastoris, paving the way for the establishment of intricate synthetic biology applications.