Optimized Isolation of Lysosome-Related Organelles from Stationary Phase and Iron-Overloaded <em>C. reinhardtii</em> Cells

Abstract

Lysosome-related organelles (LROs) are a class of heterogeneous subcellular organelles conserved in eukaryotes, performing various functions. An important function of LROs is to mediate phosphorus and metal homeostasis. Chlamydomonas reinhardtii serves as a model organism for investigating metal ion metabolism. Considering that LROs contain polyphosphate and various metal elements, the purification strategy is based on their higher density by fractionating cell lysate through OptiPrep density gradient ultracentrifugation. Here, we optimized a method for purifying LROs from C. reinhardtii cells that have reached stationary phase (sta-LROs) or are overloaded with iron (Fe-LROs). Our protocol provides technical support for further investigations on the biogenesis and function of LROs in C. reinhardtii . Key features • This protocol purifies LROs from C. reinhardtii without disrupting the structure of chloroplasts. • Following the purification of sta-LROs, these can be further fractionated into subgroups with distinct densities through the second iodixanol gradient. • This protocol is applicable for the purification of LROs from the cell wall–deficient C. reinhardtii strain cw15 .