Cloning and subcellular localization of a human phosphatidylinositol 3-phosphate 5-kinase, PIKfyve/Fab1

Abstract

Yeast Fab1 is a phosphatidylinositol 3-phosphate 5-kinase involved in endocytic membrane traffic and vacuole homeostasis. Here we have cloned and sequenced the cDNA for the human homologue of Fab1, PIKfyve. The cDNA has an open reading frame of 6294 bp and encodes a 2098-amino acid protein with a calculated molecular mass of 237 kDa, containing a phosphatidylinositol 3-phosphate-binding FYVE domain, a DEP domain, a chaperonin-like domain, and a phosphoinositide kinase domain. The human genome contains a single PIKfyve gene, which comprises 38 exons on chromosomal locus 2q34. PIKfyve is expressed as a single molecular species in a number of human cell lines derived from different tissues. The exogenously expressed protein was found to localize mainly to early endosomes containing two other FYVE domain proteins, EEA1 and Hrs. The endosomal membrane localization of PIKfyve was studied in more detail by examining cells transfected with a constitutively active mutant of the small GTPase Rab5, whose expression results in the enlargement of early endosomes. We show that PIKfyve is distributed in microdomains that are distinct from those occupied by EEA1 and Hrs.