Figure 2.

LRRK2^RCK localizes in mitochondria and at the plasma membrane. (A) Representative confocal micrographs of strains harboring endogenously green fluorescent protein (GFP)-tagged Pma1 expressing mCherry alone or fused to LRRK2^RCK and R1398L^RCK on day 1 of aging. Z-projections of three-dimensional stacks as well as a representative section are shown. Scale bar represents 5 μm. For quantification of colocalization and confocal micrographs of strains harboring GFP-tagged Htb2, please see Supplementary Figures S2A,B. (B) Representative confocal micrographs of strains harboring endogenously GFP-tagged Om45, expressing the constructs as described in (A). Z-projections of three-dimensional stacks as well as a representative section are shown. Scale bar represents 5 μm. For quantification of colocalization please see Supplementary Figure S2B. (C) Representative immunoblots of subcellular fractionation of whole cell extracts on 12%–60% step sucrose gradients. Collected fractions were analyzed by immunoblotting. Blots were probed with antibodies directed against the V5-epitope to detect V5-tagged LRRK2^RCK, and against organelle-specific marker proteins as indicated. (D) Immunoblot analysis of purified mitochondria obtained from cells expressing LRRK2^RCK or LacZ on day 1. Prior to SDS-PAGE, samples were treated with depicted concentrations of proteinase K (ProtK). Blots were probed with antibodies directed against the V5-epitope, the outer mitochondrial membrane protein Tom70, the inner mitochondrial membrane proteins Tim54, Tim22 and Tim21, the mitochondrial matrix localized protease Mas1, as well as Mcr1, located in the outer membrane (OM) and intermembrane space (IMS).