Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes

0 Department of Cell Biology, National Institute for Basic Biology , 38 Nishigonaka, Myodaijicho, Okazaki 444-8585 , Japan 1 Department of Biochemistry, The Norwegian Radium Hospital , Montebello, Oslo, 0310 , Norway 2 Department of Cell Genetics, National Institute of Genetics , Yata 1111 Mishima, Shizuoka-ken, 411-8540 , Japan 3 CREST, Japan Science and Technology Agency , Kawaguchi Center Building, 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012 , Japan 4 Department of Bioregulation and Metabolism, The Tokyo Metropolitan Institute of Medical Science , 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613 , Japan - Phosphatidylinositol-3-phosphate [PtdIns(3)P] regulates endocytic and autophagic membrane traffic. In order to understand the downstream effects of PtdIns(3)P in these processes, it is important to identify PtdIns(3)P-binding proteins, many of which contain FYVE zinc-finger domains. Here, we describe a novel giant FYVE-domaincontaining protein, named autophagy-linked FYVE protein (Alfy). Alfy is ubiquitously expressed, shares sequence similarity with the Chediak-Higashi-syndrome protein and has putative homologues in flies, nematodes and fission yeast. Alfy binds PtdIns(3)P in vitro and partially colocalizes with PtdIns(3)P in vivo. Unlike most other FYVE-domain proteins, Alfy is not found on endosomes Introduction Phosphoinositides, phosphorylated derivatives of the membrane lipid phosphatidylinositol, regulate cytoskeleton function, membrane trafficking and receptor signalling through reversible recruitment of protein complexes to specific membranes (Yin and Janmey, 2003; Toker, 2002; Simonsen et al., 2001). Phosphatidylinositol-3-phosphate [PtdIns(3)P] is formed by the phosphorylation of phosphatidylinositol by (mainly) class III phosphoinositide 3-kinases (PI-3-kinases) (Vanhaesebroeck et al., 2001). This lipid is crucial for endocytic and autophagic membrane traffic (Simonsen et al., 2001) and we are beginning to learn some of the underlying molecular mechanisms. The identification of two conserved protein domains that bind PtdIns(3)P with high specificity has facilitated the analysis of the downstream functions of this lipid. The FYVE domain is a ~70-residue zinc finger found in 27 human proteins (Stenmark et al., 2002). All FYVE domains tested to date have been found to bind to PtdIns(3)P, although a few examples that lack one or more consensus residues bind with lower affinity and also show some affinity for the structurally related phosphoinositide PtdIns(5)P (Sankaran et al., 2001). The Phox-homology (PX) domain is a ~100-residue b -sandwich but instead localizes mainly to the nuclear envelope. When HeLa cells are starved or treated with a proteasome inhibitor, Alfy relocalizes to characteristic filamentous cytoplasmic structures located close to autophagic membranes and ubiquitin-containing protein aggregates. By electron microscopy, similar structures can be found within autophagosomes. We propose that Alfy might target cytosolic protein aggregates for autophagic degradation. found in about 40 human proteins (Ellson et al., 2002). Most PX domains bind specifically to PtdIns(3)P, although some bind to other phosphoinositides. In order to learn more about how PtdIns(3)P regulates membrane trafficking, it is important to study the functions of various proteins that contain FYVE or PX domains, and several mammalian PtdIns(3)P effectors in endocytic membrane trafficking have already been identified (Simonsen et al., 1998; Raiborg et al., 2002; Nielsen et al., 2000; Ikonomov et al., 2003). Although three PtdIns(3)P effectors have been identified for the autophagy-related cytoplasm-to-vacuole targeting (Cvt) pathway in yeast (Wurmser and Emr, 2002; Nice et al., 2002), no mammalian PtdIns(3)P effectors in autophagy have been identified so far. We find it likely that such effectors exist among the many FYVE- and PX-domain-containing proteins with uncharacterized functions. In this paper, we describe a novel 400-kDa FYVE-domaincontaining protein, named autophagy-linked FYVE protein (Alfy). We show that Alfy binds to and partially colocalizes with PtdIns(3)P, and that it colocalizes with autophagic but not endocytic markers. We present evidence that Alfy might serve as a link between protein aggregates and the autophagic machinery. Materials and Methods cDNA cloning The sequence KIAA0993 [1096 nucleotides (nt)] found in the Human Unidentified Gene-Encoded Large Proteins (HUGE) database was used to design a primer (KIA-NR: 5 -GCTCCTACCTGTGAACGTGTTGACACTCA-3 ) to do a 5 rapid amplification of cDNA ends (5 -RACE) reaction with the AP-1 primer and human brain Marathon Ready cDNA as described by the manufacturer (Clontech, Palo Alto, CA), resulting in the amplification of nt 8662-9638 of Alfy. To obtain full-length Alfy cDNA (10,581 nt) several 5 -RACE reactions were performed using primers KIA315-NR (5 -CTCACGATGGACTCTGATGAATTCTCC-3 ) (nt 7796-8689), KIAcomp-NR (5 -TGCTGCATGTTCTCTTGAGTTGACTA-3 ) (nt 6597-7826), KIA-5R (5 GTATCAGTTCAGTCCAAACTCTGTTGAC-3 ) (nt 5230-6626), KIA-6 (5 -TGACAAGCATCTCGGTTAATCTCCC-3 ) (nt 43145255), KIA-7 (5 -TCTTTGCTGGCTAGTGGGTTACTCTT-3 ) (nt 3573-4340). The homologous Drosophila (AAF52302) and Caenorhabditis elegans (CAB16307,T26022,T25148) sequences were used to search The National Center for Biotechnology Information (NCBI) human genome database, and the human chromosome 4 contig RP11-147K21 (gi 8705090) was found to encompass the Alfy sequence. Homologous regions were used to design primers for PCR to generate more 5 sequence: KIA-11 (5 GAGAAACGTCCAGGCCTTTGCAGTT-3 ) + KIA-8 (5 -ATTACCAGGACCAAATGATGCCACTG-3 ) (nt 11353600) and KIA-12F (5 -CAGTTTTGTTTCCCCTGCGGAGGAGC-3 ) + KIA-10R (5 TTTCTCGAAGGACCGACAGGAGGGC-3 ) (nt 593-1912). Nucleotides 1-656 were obtained by a 5 -RACE reaction with KIA12R2 (5 -GCACTGAATAGAAGCTGGAGATCATC-3 ). The KIA12R2 RACE sequence contains a stop codon in frame, which was later confirmed by sequencing of polymerase chain reaction (PCR) products using a primer 5 to the coding region. The 5 -RACE products were cloned into the pGEM-T-easy vector (Promega, Madison, WI) and sequenced (GATC, Konstanz, Germany) using the SP6 and PCR1 primers. Plasmid constructs We were unable to subclone Alfy in one piece into a mammalian expression vector, probably because the large size of the cDNA makes it prone to recombination. We have therefore based all our immunofluorescence and electron microscopy (EM) experiments on endogenous Alfy detected using a polyclonal rabbit anti-Alfy antibody. pMAL-AlfyCT639 was generated by subcloning the Cterminal 1917 nt from Alfy into pMAL-c2 (New England Biolabs). pcDNA3-myc-Alfy2587-3527 was prepared by amplifying the relevant part of Alfy and subcloning it behind the Myc epitope of pcDNA3-myc (Raiborg et al., 2001). Human Atg5 cDNA [we here use the nomenclature by Klionsky et al. for components of the autophagic machinery (Klionsky et al., 2003)] was amplified by PCR from the human brain Ma (...truncated)