TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.

C␣-formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The FGly-generating enzyme (FGE), performing this modification, is an endoplasmic reticulum-resident enzyme that upon overexpression is secreted. Recombinant FGE was purified from cells and secretions to homogeneity. Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE. Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity. FGE is a calciumbinding protein containing an N-terminal (residues 86 -168) and a C-terminal (residues 178 -374) protease-resistant domain. The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh, and the fifth to the sixth cysteine residue. The innermost cysteine pair is partially reduced. The first two cysteine residues are located in the sequence preceding the Nterminal protease-resistant domain. They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers. The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocrosslinking of a substrate peptide to proline 182. Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species.

The recently discovered potato tuber (Solanum tuberosum) ␣-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the ␤-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx) f and Trx m from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx f being the more efficient, yielding an S0.5 value of 0.4 M. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.starch ͉ redox regulation ͉ thioredoxin

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.