The dark side of fluorescent protein tagging—the impact of protein tags on biomolecular condensation

Biomolecular condensation has emerged as an important mechanism to control various cellular processes through the formation of membraneless organelles. Fluorescent protein tags have been extensively used to study the formation and the properties of condensates in vitro and in vivo, but there is evidence that tags may perturb the condensation properties of proteins. In this study, we carefully assess the effects of protein tags on the yeast DEAD-box ATPase Dhh1, a central regulator of processing bodies (P-bodies), which are biomolecular condensates involved in mRNA metabolism. We show that fluorescent tags as well as a polyhistidine tag greatly affect Dhh1 condensation in vitro and lead to condensates with different dynamic properties. Tagging of Dhh1 with various fluorescent proteins in vivo alters the number of P-bodies upon glucose starvation and some tags even show constitutive P-bodies in nonstressed cells. These data raise concerns about the accuracy of tagged protein condensation experiments, highlighting the need for caution when interpreting the results.