Pallidin is a novel interacting protein for cytohesin‐2 and regulates the early endosomal pathway and dendritic formation in neurons

• Published in: Journal of neurochemistry 2018-10, Vol.147 (2), p.153-177
• Main Authors: Ito, Akiko, Fukaya, Masahiro, Saegusa, Shintaro, Kobayashi, Emi, Sugawara, Takeyuki, Hara, Yoshinobu, Yamauchi, Junji, Okamoto, Hirotsugu, Sakagami, Hiroyuki
• Format: Article
• Language: English
• Subjects: Actin; Adenosine diphosphate; ADP ribosylation factor; BLOC‐1; Brain; dendrite; Dendritic structure; endosome; Endosomes; Glutathione; Guanine; Guanine nucleotide exchange factor; Hippocampus; Immunoelectron microscopy; Immunofluorescence; Immunoprecipitation; Membrane trafficking; Membrane vesicles; Microscopy; Neurons; Organelles; Proteins; protein‐protein interactions; Syntaxin; Yeast
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/jnc.14579

Cytohesin‐2 is a member of the guanine nucleotide exchange factors for ADP ribosylation factor 1 (Arf1) and Arf6, which are small GTPases that regulate membrane traffic and actin dynamics. In this study, we first demonstrated that cytohesin‐2 localized to the plasma membrane and vesicles in various subcellular compartment in hippocampal neurons by immunoelectron microscopy. Next, to understand the molecular network of cytohesin‐2 in neurons, we conducted yeast two‐hybrid screening of brain cDNA libraries using cytohesin‐2 as bait and isolated pallidin, a component of the biogenesis of lysosome‐related organelles complex 1 (BLOC‐1) involved in endosomal trafficking. Pallidin interacted specifically with cytohesin‐2 among cytohesin family members. Glutathione S‐transferase pull‐down and immunoprecipitation assays further confirmed the formation of a protein complex between cytohesin‐2 and pallidin. Immunofluorescence demonstrated that cytohesin‐2 and pallidin partially colocalized in various subsets of endosomes immunopositive for EEA1, syntaxin 12, and LAMP2 in hippocampal neurons. Knockdown of pallidin or cytohesin‐2 reduced cytoplasmic EEA1‐positive early endosomes. Furthermore, knockdown of pallidin increased the total dendritic length of cultured hippocampal neurons, which was rescued by co‐expression of wild‐type pallidin but not a mutant lacking the ability to interact with cytohesin‐2. In contrast, knockdown of cytohesin‐2 had the opposite effect on total dendritic length. The present results suggested that the interaction between pallidin and cytohesin‐2 may participate in various neuronal functions such as endosomal trafficking and dendritic formation in hippocampal neurons. Cover Image for this issue: doi: 10.1111/jnc.14197. Cytohesin‐2 is a guanine nucleotide exchange factor that is implicated in various neuronal functions through the activation of ADP ribosylation factors 1 (Arf1) and Arf6. In this study, we identified pallidin as a novel interacting protein specific for cytohesin‐2 among the cytohesin family. In cultured hippocampal neurons, knockdown of either cytohesin‐2 or pallidin similarly affected the endocytic pathway. Furthermore, knockdown of pallidin had a stimulatory effect on dendritic extension via its coiled coil domain that was responsible for the interaction with cytohesin‐2. These results provide the first evidence for a functional relationship between cytohesin‐2 and pallidin in neurons. Cover Image for this issue: doi: 10.111