Protocol for the colocalization of yeast peroxisomal membrane proteins and their binding partners using stimulated emission depletion microscopy

Peroxisomes are highly dynamic organelles that play crucial roles in cellular metabolism. Here, we present a protocol to investigate peroxisomal membrane proteins and their binding partners in the yeast Hansenula polymorpha using stimulated emission depletion (STED) nanoscopy. We describe steps for strain construction to facilitate specific STED-compatible labeling, automated STED imaging, and semi-automated data analysis. This protocol enables examination of the dynamic alterations in the colocalization of the peroxisomal membrane protein Pex3 and its binding partner Atg30. For complete details on the use and execution of this protocol, please refer to de Lange et al.¹.