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Alix (also named AIP1) is an interacting partner of the penta-EF-hand Ca 2 + -binding protein, ALG-2 [1-5], and acts as a multifunctional adaptor protein in various cellular functions such as cell death, receptor endocytosis, endosomal protein sorting, cell adhesion, budding of enveloped RNA viruses and development 10.1111/j.1742-4658.2007.06230.x Human Brox is a newly identified 46 kDa protein that has a Bro1 domainlike 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 Tac , 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 FLAGCHMP4b. 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.Abbreviations Brox WT , wild-type Brox; CAAX motif, a thioester-linkage site of isoprenoid lipid (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid); CHMP, charged multivesicular body protein; E-64, trans-epoxysuccinyl-L-leucylamido(4-guanidino) butane; ESCRT, endosomal sorting complex required for transport; FTI-27, farnesyltransferase inhibitor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione S-transferase; mGFP, monomeric green fluorescent protein; pAb, polyclonal antibody; PTP, protein tyrosine phosphatase; PVDF, poly(vinylidene difluoride); RabGAPLP, Rab GTPase-activating protein-like protein; Strep, StrepTactin binding sequence.

†These authors contributed equally to this work.Regulator of ubiquitous kinase/Cbl-interacting protein of 85 kDa (Ruk/CIN85) and CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3-kinase (PI 3-kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI-coated vesicles involved in retrograde endoplasmic reticulum-Golgi and intra-Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin-mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.

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