Abstract The objective of the research proposal is to obtain a molecular understanding of the phosphomannosyl targeting system which functions to deliver newly synthesized acid hydrolases to lysosomes. Defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases. The specific aims include: (1) Characterization of the protein recognition domain on DNase I that is necessary for interaction with phosphotransferase which catalyzes the first step in the generation of the Man-6-P recognition marker. We will use site-directed mutagenesis to identify residues on the surface of DNase I that determine binding to phosphotransferase and analyze how these residues direct phosphorylation at selected glycosylation sites. (2) Identification of amino acids in the cytoplasmic tail of phosphodiester alpha-GlcNAcase that serve as signals for its trafficking between the trans-Golgi network (TGN) and the plasma membrane. This enzyme removes the covering GlcNAc from acid hydrolase oligosaccharides to expose Man-6-P residues that mediate binding to Man-6-P receptors. GST peptides with these amino acids will be used to isolate proteins that recognize these motifs. Antibodies to the cytoplasmic tail will used to immunoisolate the TGN for characterization of its protein composition. (3) Analysis of how phosphorylation/dephosphorylation events regulate the interaction of the GGA proteins with the Man-6-P receptors and the coat protein AP-1. Recent evidence shows that the GGAs bind Man-6-P receptors in the Golgi and present then to AP-1 for packaging into clathrin-coated vesicles. We will (a) define the amino acids in the hinge segments of the GGAs that bind to the ear domain of the gamma subunit of AP-1. (b) determine how phosphorylation of GGAs 1, 3 by AP-1 associated casein kinase 2 alters GGA protein conformation resulting in dissociation from AP-1 and the release of bound Man-&P receptors. (c) identify the phosphatase that acts on GGAs 1,3. A candidate is protein phosphatase 2a that is known to bind to the MPR cytoplasmic tail. (d) perform in vitro assays to determine whether the GGAs can nucleate clathrin coated vesicles in the absence of AP-1. This addresses the issue of whether GGAs form their own transport vesicles in addition to serving as accessory proteins for AP-1 vesicle assembly. (4) Studies to identify the cytosolic proteins required for AP-1 recruitment onto liposomes will be continued. Research Plan for the Extension (1) A major goal of our work will be to further our understanding of the interactions of the GGA proteins with the MPRs and AP-1. Our findings to date indicate that phosphorylation / dephosphorylation of GGAs 1 and 3 regulate these processes. We have found that AP-I contains tightly bound casein kinas2e (CK-2) that phosphorylates GGAs 1/3. We will identify which subunit of the tetrameric AP-1 binds CK-2b?? digesting the molecule with trypsin to selectively cleave the hinge region of the s61 and ?? subunits and determining if the CK-2 is released or remains with the trunk portion of the AP-1. Guided by this result we will perform GST-pull down experiments using appropriate domains of AP-1 to pull down soluble CK-2. Next we will identify the phosphatase that dephosphorylates GGAs113, thereby allowing these molecules to bind to the MPRs and AP-1. A candidate is protein phosphatase 2a (PP2a) that is known to bind to several Golgi proteins including the MPR. With CK-2 and the phosphatase in hand, the effect of phosphorylation on the conformation of GGAs 1/3 will be studied by gel filtration, sucrose density gradients and protease sensitivity. These reagents will also allow us to confirm our preliminary data that phosphorylation of GGAs 1/3 impairs binding to the AP-1 ?? subunit, thereby providing a mechanism for the dissociation of this complex. To better understand the GGA-AP-1 interaction,w e will use mutagenesis to identify the amino acids of the GGA hinge domain that interact with the AP-I ?? ear. We will also determine whether or not the GGA hinge binds to the same surface of the AP-1 ?? ear as ?? synergin and rabaptin 5, two proteins known to bind to the ?? ear. These experiments will be guided by the recently published ?? ear structure. In collaboration with Hans Geuze, we will use immuno E/M to analyze the subcellular distributions of GGAl molecules that have serine 355 mutated to alanine (to prevent phosphorylation)o r to aspartate (to mimic phosphorylation). We postulate that both mutants will be recruited onto the TGN, but the latter will be incapable of binding to MPRs or AP-1 and therefore its localization into AP-I containing clathrin-coated buds will be impaired. These studies will help to correlate our in vitro findings with the behavior of the GGAs in intact cells. Finally, we will study the recruitment of purified GGAs onto Golgi membranes and liposomes to determine whether GGAs can nucleate CCVs. While GGAs are known to bind clathrin, there is no evidence that they nucleate CCVs. This is an important issue in regards to whether the GGAs form CCVs independent of their interaction with AP-1. (2) We will continue to define the protein recognition domain on DNase I that allows binding to phosphotransferase. This interaction is central to understanding how acid hydrolases are selectively phosphorylated. Our findings to date indicate that lysine residues are only one component of the binding surface. By using non-glycosylated DNase I mutants expressed in bacteria to inhibit phosphorylation of intact acid hydrolases by phosphotransferase, we hope to identify the other amino acids that are involved in binding. The availability of mg amounts of recombinant phosphotransferase (provided by Dr. William Canfield at Novazyme) will allow us for the first time to perform direct binding studies between this enzyme and its protein substrates. (3) We will extend our studies of !?Uncovering Enzyme!? trafficking by identifying the amino acid residues in the cytoplasmic tail of UCE that allow exit from the TGN. Once these are defined, we will use a GST-fusion protein containing these residues in pull-down assays to search for an interacting protein(s). Using a similar approach we will try to identify a protein(s) that binds to the 486-Ycontaining motif that retains UCE in the endosome for return to the TGN. Identification of such proteins will advance our understanding of how the trafficking of UCE is regulated. We have prepared high affinity antibodies to the cytoplasmic tail of UCE for use in the immuno isolation of the TGN (where the bulk of UCE is localized). If successful this would be the first immunoisolation of a specific Golgi compartment. We will identify the protein components by 2-D gel analysis, FABOMass Spectrometry and western blotting using antibodies to candidate proteins. New proteins will be cloned and their localization in the TGN confirmed by immuno fluorescence. These experiments may identify novel functions of the TGN. (4) We will pursue the purification of the cytosolic protein required for AP-1 recruitment onto liposomes. If successful, we will obtain amino acid sequence to determine if it is a known protein. If it is novel, we will clone it to obtain its sequence and identify protein domains that may give clues as to its function. We will then study how the protein facilitates AP-1 recruitment.