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Brox, a novel farnesylated Bro1 domain‐containing protein that associates with charged multivesicular body protein 4 (CHMP4)
Author(s) -
Ichioka Fumitaka,
Kobayashi Ryota,
Katoh Keiichi,
Shibata Hideki,
Maki Masatoshi
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2007.06230.x
Subject(s) - hek 293 cells , mutant , transport protein , microbiology and biotechnology , biology , biochemistry , receptor , gene
Human Brox is a newly identified 46 kDa protein that has a Bro1 domain‐like sequence and a C‐terminal thioester‐linkage site of isoprenoid lipid (CAAX motif) (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid). Mammalian Alix and its yeast ortholog, Bro1, are known to associate with charged multivesicular body protein 4 (CHMP4), a component of endosomal sorting complex required for transport III, via their Bro1 domains and to play roles in sorting of ubiquitinated cargoes. We investigated whether Brox has an authentic Bro1 domain on the basis of its capacity for interacting with CHMP4s. Both Strep Tactin binding sequence (Strep)‐tagged wild‐type Brox (Strep–Brox WT ) and Strep‐tagged farnesylation‐defective mutant (Cys→Ser mutation; Strep–Brox C408S ) pulled down FLAG‐tagged CHMP4b that was coexpressed in HEK293 cells. Treatment of cells with a farnesyltransferase inhibitor, FTI‐277, caused an electrophoretic mobility shift of Strep–Brox WT , and the mobility coincided with that of Strep–Brox C408S . The inhibitor also caused a mobility shift of endogenous Brox detected by western blotting using polyclonal antibodies to Brox, suggesting farnesylation of Brox in vivo . Fluorescence microscopic analyses revealed that Strep–Brox WT exhibited accumulation in the perinuclear area and caused a punctate pattern of FLAG–CHMP4b that was constitutively expressed in HEK293 cells. On the other hand, Strep–Brox C408S showed a diffuse pattern throughout the cell, including the nucleus, and did not cause accumulation of FLAG–CHMP4b. Fluorescent signals of monomeric green fluorescent protein (mGFP)‐fused Brox WT merged partly with those of Golgi markers and with those of abnormal endosomes induced by overexpression of a dominant negative mutant of AAA type ATPase SKD1/Vps4B in HeLa cells, but such colocalization was less efficient for mGFP–Brox C408S . These results suggest a physiological significance of farnesylation of Brox in its subcellular distribution and efficient interaction with CHMP4s in vivo .