The steps leading to constitutive exocytosis are poorly understood. In Dictyostelium WASH complex mutants, exocytosis is blocked, so cells that take up fluorescent dextran from the medium retain it and remain fluorescent. Here we establish a FACS-based method to select cells that retain fluorescent dextran, allowing identification of mutants with disrupted exocytosis. Screening a pool of random mutants identified the WASH complex, as expected, and multiple mutants in the conserved HEAT-repeat containing protein Mroh1. In mroh1 mutants, endosomes develop normally until the stage where lysosomes neutralize to postlysosomes, but thereafter the WASH complex is recycled inefficiently, and subsequent exocytosis is substantially delayed. Mroh1 protein localizes to lysosomes in mammalian and Dictyostelium cells. In Dictyostelium it accumulates on lysosomes as they mature, and is removed together with WASH shortly before the postlysosomes are exocytosed. WASH-generated F-actin is required for correct subcellular localisation; in WASH complex mutants, and immediately after latrunculin treatment, Mroh1 relocalises from cytoplasm to small vesicles. Thus Mroh1 is involved in a late and hitherto undefined actin-dependent step in exocytosis.
- Received September 12, 2016.
- Accepted March 29, 2017.
- © 2017. Published by The Company of Biologists Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.