Promotion of neurite extension by protrudin requires its interaction with vesicle-associated membrane protein-associated protein.
Protrudin is a protein that contains a Rab11-binding domain and a FYVE (lipid-binding) domain and that functions to promote neurite formation through interaction with the GDP-bound form of Rab11. Protrudin also contains a short sequence motif designated FFAT (two phenylalanines in an acidic tract), which in other proteins has been shown to mediate binding to vesicle-associated membrane protein-associated protein (VAP). We now show that protrudin associates and colocalizes with VAP-A, an isoform of VAP expressed in the endoplasmic reticulum. Both the interaction between protrudin and VAP-A as well as the induction of process formation by protrudin were markedly inhibited by mutation of the FFAT motif. Furthermore, depletion of VAP-A by RNA interference resulted in mislocalization of protrudin as well as in inhibition of neurite outgrowth induced by nerve growth factor in rat pheochromocytoma PC12 cells. These defects resulting from depletion of endogenous rat VAP-A in PC12 cells were corrected by forced expression of (RNA interference-resistant) human VAP-A but not by VAP-A mutants that have lost the ability to interact with protrudin. These results suggest that VAP-A is an important regulator both of the subcellular localization of protrudin and of its ability to stimulate neurite outgrowth.
Saita
Shotaro
S
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan.
Shirane
Michiko
M
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan.
Natume
Tohru
T
National Institutes of Advanced Industrial Science, Kohtoh-ku, Tokyo 135-0064, Japan.
Iemura
Shun-Ichiro
SI
National Institutes of Advanced Industrial Science, Kohtoh-ku, Tokyo 135-0064, Japan.
Nakayama
Keiichi I
KI
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan. Electronic address: nakayak1@bioreg.kyushu-u.ac.jp.
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D016501:Neurites / Q000378:metabolism*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D002352:Carrier Proteins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C114051:VAPA protein, human; C513390:ZFYVE27 protein, human; C514782:Zfyve27 protein, rat