title: LRRK2 transport is regulated by its novel interacting partner Rab32.
abstract: Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2.
authors.affiliation: Department of Experimental Tumorbiology, Westfälische Wilhelms University Muenster, Muenster, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Waschbüsch
authors.affiliation: Department of Experimental Tumorbiology, Westfälische Wilhelms University Muenster, Muenster, Germany.
authors.forename: Helen
authors.initials: H
authors.lastname: Michels
authors.affiliation: Department of Experimental Tumorbiology, Westfälische Wilhelms University Muenster, Muenster, Germany.
authors.forename: Swantje
authors.initials: S
authors.lastname: Strassheim
authors.affiliation: Department of Experimental Tumorbiology, Westfälische Wilhelms University Muenster, Muenster, Germany.
authors.forename: Edith
authors.initials: E
authors.lastname: Ossendorf
authors.affiliation: Department of Experimental Tumorbiology, Westfälische Wilhelms University Muenster, Muenster, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kessler
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum München, Neuherberg, Germany; Medical Proteome Center, Institute for Ophthalmic Research, Eberhard Karls University Tübingen, Tübingen, Germany.
authors.forename: Christian Johannes
authors.initials: CJ
authors.lastname: Gloeckner
authors.affiliation: Department of Experimental Tumorbiology, Westfälische Wilhelms University Muenster, Muenster, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Barnekow
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002451:Cell Compartmentation; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D042541:Intracellular Space / Q000378:metabolism; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D050505:Mutant Proteins; D049452:Green Fluorescent Proteins; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; C466094:Rab32 protein, human; D020691:rab GTP-Binding Proteins