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Oligomerization of ZFYVE27 (Protrudin) is necessary to promote neurite extension.

PloS one (2012-01-05)
D V Krishna Pantakani, Marta M Czyzewska, Anna Sikorska, Chiranjeevi Bodda, Ashraf U Mannan
ABSTRACT

ZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27's self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27(ΔHR3) failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27(ΔHR3) was co-expressed with wild-type ZFYVE27 (ZFYVE27(WT)), it exerted a dominant negative effect on ZFYVE27(WT) as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
IGEPAL® CA-720, average Mn ~735
Sigma-Aldrich
Triton X-114
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IGEPAL® CA-630, viscous liquid
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IGEPAL® CA-630, for molecular biology
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Triton X-165 solution
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Triton X-114, laboratory grade
Supelco
IGEPAL® CA-630
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Anti-ZFYVE27 (AB1) antibody produced in rabbit, IgG fraction of antiserum
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Triton X-405 solution, 70% in H2O