The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate beta1,4-galactosyltransferase 1 (galT) and alpha2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT-siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.

as the key residue) in its cytoplasmic tail is required for the sorting of the receptor from late endosomes back to the Golgi apparatus. However, the cationindependent mannose 6-phosphate receptor (CI-MPR) lacks such a di-aromatic motif. Therefore the ability of amino acids other than aromatic residues to replace Trp 19 in the CD-MPR cytoplasmic tail was tested. Mutant constructs with bulky hydrophobic residues (valine, isoleucine, or leucine) instead of Trp 19 exhibited 30 -60% decreases in binding to the tail interacting protein of 47 kDa (Tip47), a protein mediating this transport step, and partially prevented receptor delivery to lysosomes. Decreasing hydrophobicity of residues at position 19 resulted in further impairment of Tip47 binding and an increase of receptor accumulation in lysosomes. Intriguingly, mutants mislocalized to lysosomes did not completely co-localize with a lysosomal membrane protein, which might suggest the presence of subdomains within lysosomes. These data indicate that sorting of the CD-MPR in late endosomes requires a distinct di-aromatic motif with only limited possibilities for variations, in contrast to the CI-MPR, which seems to require a putative loop (Pro 49 -Pro-Ala-Pro-Arg-Pro-Gly 55 ) along with additional hydrophobic residues in the cytoplasmic tail. This raises the possibility of two separate binding sites on Tip47 because both receptors require binding to Tip47 for endosomal sorting.

IntroductionLysosomes are one of the major degradative compartments of mammalian cells and their biogenesis depends on the concerted transport of acid hydrolases from their site of origin to their site of action through a series of intermediate compartments. Lysosomal enzymes are recognized by one of the two known mannose 6-phosphate receptors (MPRs) in the trans-Golgi network (TGN) for transport onward to lysosomes. This crucial recognition event directly depends on the acquisition of mannose 6-phosphate (M6P) tags by the lysosomal enzymes. The tag is generated in a two-step enzymatic reaction which occurs in the Golgi. The first step, the addition of an N-acetyl glucosamine phosphate to selected mannose residues of the lysosomal enzyme, is catalyzed by the UDP-N-acetylglucosamine lysosomal enzyme, N-acetylglucosamine phosphotransferase. The second step, mediated by N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase, leads to the actual uncovering of the M6P tag by cleaving the GlcNAc residue. The α-N-acetylglucosaminidase is therefore termed uncovering enzyme (UCE).UCE is a type I membrane glycoprotein of 515 amino acids. It has a single transmembrane domain of 27 residues and a 41 residue cytoplasmic tail, which contains several established and potential signal sequences ensuring its correct intracellular sorting (Kornfeld et al., 1999;Lee et al., 2002). UCE has been shown to reside in the TGN at steady state and its constitutive recycling between that organelle and the plasma membrane has been established using a combination of steady state measurements of enzyme activity and localization of green fluorescent protein (GFP) fusion constructs of the enzyme cytoplasmic tail (Rohrer and Kornfeld, 2001). It was shown that the classical endocytosis motif Y 488 HPL and the Cterminal N 511 PFKD 515 motif are involved in the trafficking of UCE (Rohrer and Kornfeld, 2001). However, the dissection of the trafficking itinerary of UCE to elucidate the individual transport steps involved has not yet been reported. Three different types of signals in the cytoplasmic tail of UCE have been described. First, the transmembrane domain and the first 11 residues of the cytoplasmic tail of UCE were shown to be involved in TGN retention of the enzyme, thereby indicating 2949The human mannose 6-phosphate uncovering enzyme participates in the uncovering of the mannose 6-phosphate recognition tag on lysosomal enzymes, a process that facilitates recognition of those enzymes by mannose 6-phosphate receptors to ensure delivery to lysosomes. Uncovering enzyme has been identified on the trans-Golgi network at steady state. It has been shown to traffic to the plasma membrane from where it is rapidly internalized via endosomal structures, the process being mediated by a tyrosine-based internalization motif, Y 488 HPL, in its cytoplasmic tail. Using immunogold electron microscopy a GFP-uncovering enzyme fusion construct was found to be colocalized with the cation-dependent mannose 6-phosophate receptor in regions of the trans-Golg...

Lysosomes are membrane-bound degradative organelles that are found in all higher eukaryotic cells. The limiting membranes of lysosomes contain a characteristic set of transmembrane glycoproteins that are transported to lysosomes by virtue of lysosomal targeting motifs found in the proteins' cytoplasmic tails (Bonifacino and Dell'angelica, 1999) The lumens of lysosomes contain an assortment of acidic hydrolases. These enzymes are synthesized in the endoplasmic reticulum (ER), where they become core-glycosylated. They are then transported into the Golgi complex, where they acquire a mannose-6-phosphate recognition tag in a two-step enzymatic process mediated by a phosphotransferase and an uncovering enzyme. The tag is then recognized by cation-dependent and cation-independent mannose-6-phoshate receptors in the trans-Golgi network (TGN). The resulting enzyme-receptor complexes are transported onward to endosomes, where the acidic pH induces dissociation of the enzymes from the receptors. The enzymes are then transported to lysosomes, while the receptors recycle back to the TGN to facilitate further rounds of transport. Failure of the receptors to recycle results in their misrouting to (and subsequent degradation in) lysosomes. The return of these receptors from endosomes to the TGN depends on specific amino acid residues (including specific cysteine residues that undergo reversible palmitoylation) in the cytoplasmic tails of receptor molecules. The final delivery of lysosomal enzymes to lysosomes has been proposed to occur by a fusion-fission process, referred to as "kiss and run," involving late endosomes and lysosomes (Luzio et al., 2003). BASIC PROTOCOL FRACTIONATION OF CELLS USING A SELF-FORMING PERCOLL DENSITY GRADIENT Sedimentation on self-forming Percoll gradients can be used to separate dense lysosomes from lower-density organelles such as endosomes, Golgi apparatus, plasma membrane, and ER. Separation is based on the sedimentation of particles to their isopycnic positioni.e., the position where the gradient density is equal to the density of the particle. The organelles are thus separated solely on the basis of differences in density, irrespective of their size. The gradient described in this protocol is a continuous gradient that forms

Abstract. Two major functions of the Golgi apparatus (GA) are formation of complex glycans and sorting of proteins destined for various subcellular compartments or secretion. To fulfill these tasks proper localization of the accessory proteins within the different sub-compartments of the GA is crucial. Here we investigate structural determinants mediating transition of the two glycosyltransferases b-1,4-galactosyltransferase 1 (gal-T1) and the a-1,3-fucosyltransferase 6 (fuc-T6) from the trans-Golgi cisterna to the trans-Golgi network (TGN). Upon treatment with the ionophore monensin both glycosyltransferases are found in TGN-derived swollen vesicles, as determined by confocal fluorescence microscopy and density gradient fractionation. Both enzymes carry a signal consisting of the amino acids E 5 P 6 in gal-T1 and D 2 P 3 in fuc-T6 necessary for the transition of these glycosyltransferases from the trans-Golgi cisterna to the TGN, but not for their steady state localization in the trans-Golgi cisterna.