SUMMARY Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends upon PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy.

The sorting of transmembrane proteins (e.g., cell surface receptors) into the multivesicular body (MVB) pathway to the lysosomal/vacuolar lumen requires the function of the ESCRT protein complexes. The soluble coiled-coil-containing proteins Vps2, Vps20, Vps24, and Snf7 are recruited from the cytoplasm to endosomal membranes where they oligomerize into a protein complex, ESCRT-III. ESCRT-III contains two functionally distinct subcomplexes. The Vps20-Snf7 subcomplex binds to the endosomal membrane, in part via the myristoyl group of Vps20. The Vps2-Vps24 subcomplex binds to the Vps20-Snf7 complex and thereby serves to recruit additional cofactors to this site of protein sorting. We provide evidence for a role for ESCRT-III in sorting and/or concentration of MVB cargoes.

A novel MVB/lysosomal sorting pathway for signaling receptors bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be broadly applicable to GPCRs containing YPXnL motifs.

The GTPase Rab1 regulates endoplasmic reticulum-Golgi and early Golgi traffic. The guanine nucleotide exchange factor (GEF) or factors that activate Rab1 at these stages of the secretory pathway are currently unknown. Trs130p is a subunit of the yeast TRAPPII (transport protein particle II) complex, a multisubunit tethering complex that is a GEF for the Rab1 homologue Ypt1p. Here, we show that mammalian Trs130 (mTrs130) is a component of an analogous TRAPP complex in mammalian cells, and we describe for the first time the role that this complex plays in membrane traffic. mTRAPPII is enriched on COPI (Coat Protein I)-coated vesicles and buds, but not Golgi cisternae, and it specifically activates Rab1. In addition, we find that mTRAPPII binds to gamma1COP, a COPI coat adaptor subunit. The depletion of mTrs130 by short hairpin RNA leads to an increase of vesicles in the vicinity of the Golgi and the accumulation of cargo in an early Golgi compartment. We propose that mTRAPPII is a Rab1 GEF that tethers COPI-coated vesicles to early Golgi membranes.

R egulated targeting of proteins to appropriate sites of action within cells is accomplished in part by enzymatic modification of the address site that is recognized by modular protein domains. Phox (PX) domains are Ϸ120-residue protein modules initially recognized by sequence homology in NADPH oxidase subunits, sorting nexins, and PI 3-kinases (1). Proteins containing PX domains are associated with specific membrane compartments, and the PX domains of Vam7p, p40 Phox , p47 Phox , CISK, and sorting nexin (SNX) 3 recognize specific phosphatidylinositol (PtdIns) (2-8). All PX domains of yeast are reported to recognize PtdIns (3) P (9). Binding affinities of the yeast proteins, however, differ up to 10 3 -fold in vitro, suggesting that membrane association in vivo depends on additional protein⅐protein interactions (9). SNX1, initially identified via its interaction with epidermal growth factor receptor (EGFR), contains a PX domain in its NH 2 terminus and three predicted coiled-coils in its COOH terminus (10). Other SNX family members also contain coiledcoils (SNX2 and SNX4), contain little sequence beyond a PX domain (SNX3), or contain various functional domains (an RGS domain in SNX13͞RGS-PX1, an SH3 domain in SNX9) (11)(12)(13)(14). hSNX1 interacts with orthologs of three yeast proteins that together with Vp5p, the yeast ortholog of SNX1, form the retromer complex, suggesting conservation of function in vesicular trafficking in the endocytic system (15, 16).We have analyzed the role of the PX and coiled-coil domains of SNX1 in subcellular localization to tubulovesicular structures and in interactions with SNX family members. We assessed the effects of regulated overexpression of the NH 2 terminus of SNX1 that contains the PX domain on ligand-induced down-regulation of EGFR. The results of these studies indicate that both an intact PX domain that recognizes PtdIns lipids and the coiled-coil domains of SNX1 involved in protein⅐protein interactions are required for proper vesicle membrane targeting. Neither domain alone is sufficient. We conclude that SNX1 functions to enhance EGFR trafficking in the endosome to lysosome pathway. Materials and MethodsRabbits were immunized with the purified PX domain of SNX1 (aa 142-269) and the resultant antisera that was affinity purified (8501) specifically recognized SNX1 but not SNX2 or SNX3. The SEFIGA rabbit polyclonal antibody was generated using a peptide corresponding to the COOH terminus of hEGFR.Protein Expression, Purification, and Characterization. Sequences encoding PX domains were amplified in PCR reactions from cDNAs encoding the indicated proteins, digested using appropriate restriction enzymes and cloned into expression vectors. Mutations were prepared using oligonucleotide-directed mutagenesis and verified by sequencing. Proteins were expressed in Escherichia coli and soluble proteins were purified on the appropriate affinity columns (NTA, Glutathione, or Chitin agarose) and chromatographed on a Superdex S200 column. Crosslinking of the SNX1 PX domain was carrie...