A luminal proteome of the endoplasmic reticulum and Golgi apparatus reveals a novel modulator of ER stress tolerance in African trypanosomes.

African trypanosomes employ specialized mechanisms of membrane trafficking as a strategy to persist in both the mammalian host and insect vector. Their survival relies on continuous synthesis and surface delivery of extremely abundant surface coat proteins, imposing an extraordinary biosynthetic burden on the secretory pathway. Despite this, their ER and Golgi luminal proteomes remain incompletely characterized. Here, we exploit proximity biotinylation, using the abundant ER chaperone BiP as bait to map the ER proteome in bloodstream and procyclic lifecycle stages. Comparison with BiPN, a truncated secretory form of BiP that escapes the ER and transits the Golgi, provides differential compartmental labelling. Quantitative ranking of BiP labelling intensity identifies a candidate BiP interactor cohort including a divergent homologue to the mammalian BiP nucleotide exchange inhibitor MANF. Reciprocal manipulation of TbMANF abundance produces opposing shifts in cellular sensitivity to ER stress. These data are consistent with a role in regulating BiP ATPase cycling in an organism that, unlike opisthokonts, lacks a canonical unfolded protein response, making TbMANF the first regulator of BiP activity identified in kinetoplastids. Finally, proximity labelling anchored at the inner face of the nuclear pore via NUP65 extends our endomembrane map to the inner nuclear membrane, identifying candidate proteins of this specialized ER-continuous domain.