No abstract

No abstract

Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep Gz cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin.MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes.When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well.Mannnose 6-phosphate receptors (MPR) 1 mediate the selective targeting of newly synthesized lysosomal enzymes to lysosomes. The receptors recognize mannose 6-phosphate residues which are added to the nascent enzyme molecules in, or in association with, the Golgi complex (1). After receptor-ligand binding, the complexes travel via an unknown pathway to the lysosomes. Before enzyme delivery, MPR and ligands uncouple in the acidic internal milieu of some prelysosomal compartment. Uncoupling permits free receptors to be re-used (2). The idea of recycling MPR stems mainly from studies on enzyme uptake by cells. These studies also indicate Abbreviations used in this paper. ASGP, asialoglycoprotein; CHO, Chinese hamster ovary; CURL, compartment of uncoupling receptors and ligands; GERL, region of smooth endoplasmic retieulum at the inner or trans-face of the Golgi stack; MPR, mannose 6-phosphate receptors; mvb's, multivesicular bodies; TGR, trans-Golgl reticulum.

We searched for novel proteins in lysosomal membranes, tentatively participating in molecular transport across the membrane and/or in interactions with other compartments. In membranes purified from placental lysosomes, we identified 58 proteins, known to reside at least partially in the lysosomal membrane. These included 17 polypeptides comprising or associated with the vacuolar adenosine triphosphatase. We report on additional 86 proteins that were significantly enriched in the lysosomal membrane fraction. Among these, 12 novel proteins of unknown functions were found. Three were orthologues of rat proteins that have been identified in tritosomes by Bagshaw RD et al. (A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle. Mol Cell Proteomics 2005;4:133-143). Here, the proteins encoded by LOC201931 (FLJ38482) and LOC51622 (C7orf28A) were expressed with an appended fluorescent tag in HeLa cells and found to be present in lysosomal organelles. Among the lysosomally enriched proteins, also 16 enzymes and transporters were detected that had not been assigned to lysosomal membranes previously. Finally, our results identified a particular set of proteins with known functions in signaling and targeting to be at least partially associated with lysosomes.

Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from co-purifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.

Carboxypeptidase Y, a vacuolar enzyme in Saccharomyces cerevuia, is synthesized as a larger precursor whose apparent molecular mass is approximately 67,000 daltons. We have characterized a recessive mutation, pep4-3, that prevents maturation of this precursor. The accumulated precursor does not possess enzymatic activity. We have shown that the precursor accumulating in the pep4-3 mutant is not produced in a doubly mutant strain that also bears a mutation in the carboxypeptidase Y structural gene that eliminates production of carboxypeptidase Y. We have also shown that a nonsense fragment of carboxypeptidase Y is processed. Although there is evidence that proteinase B can catalyze the conversion of the precursor to a mature form in vitro, nonsense mutations in the structural gene for proteinase B, PRBI, do not affect the levels of carboxypeptidase Y activity, and strains bearing these mutations produce a carboxypeptidase Y of apparently normal size. Hence, proteinase B is not essential for the maturation of carboxypeptidase Y precursor in viva The pep4-3 mutation affects at least five vacuolar enzymes. This suggests that there is a processing event common to all of these enzymes.The posttranslational cleavage of peptide fragments from precursor proteins has been shown to play a crucial role in the assembly of viral capsids (1) and collagen molecules (2), in the activation of various proenzymes (3, 4) and prohormones (5-15), and in the cascade of events that leads to coagulation (16,17) and complement fixation (18) in blood. In addition, the scission of a hydrophobic amino-terminal segment from secreted proteins has been hypothesized to be an integral component of the process that results in the cellular localization of proteins (19,20). The observation that many secreted proteins contain an oligosaccharide moiety has invited speculation concerning the importance of this carbohydrate to the secretion process (21,22).The yeast Saccharomyces cerevisiae synthesizes a spectrum of proteins that become localized either within the vacuole or outside of the plasma membrane. In yeast cells the vacuole is a prominent organelle and it contains a variety of hydrolytic enzymes, amongst them proteinase A (EC 3.4.23.6), proteinase B (EC 3.4.22.9), and carboxypeptidase Y (23-27).Carboxypeptidase Y is a glycoprotein with a molecular mass of about 61,000 daltons, of which approximately 10,000 daltons is contributed by the oligosaccharide (28-34). Hasilik and Tanner have shown that carboxypeptidase Y is synthesized as a 67,000-dalton precursor, and that this precursor contains mannose. The maturation of the precursor occurs proteolytically and the same or a very similar cleavage can be catalyzed by proteinase B or trypsin, in vitro (35,36).Sixteen genes have been described whose function is essential for the production of carboxypeptidase Y in yeast (37). Of these, PRCI is the structural gene for carboxypeptidase Y (38). Mutations at the PEP4 locus are recessive and pleiotropic (37). The pep4-3 mutation reduces proteinase ...

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-#-N-acetylglucosaminidase H. After a 10-min pulse, It is now well established that cathepsin D is synthesized on membrane-bound ribosomes and cotranslationally translocated into the lumen of the endoplasmic reticulum (1-3). Cathepsin D synthesized in human skin fibroblasts contains two asparagine-linked oligosaccharides per polypeptide chain. During passage through the Golgi apparatus, a portion of these oligosaccharides may become phosphorylated or converted into complex-type oligosaccharides. In the presence of NH4CI, ~90% of the newly synthesized cathepsin D is secreted. In the NH4Cl-induced secretions, about half of the cathepsin D molecules have one high-mannose and one complex oligosaccharide. The remaining cathepsin D polypeptides have either two high-mannose or two complex oligosaccharides (4). Of the high-mannose oligosaccharides, about half are phosphorylated (5). Subcellular fractionation experiments (6) and kinetic studies (7) established that phosphorylation represents an early reaction in the Golgi apparatus and precedes the formation of complex oligosaccharides. The segregation of cathepsin D and of other lysosomal enzymes from the secretory pathway is dependent on binding to mannose 6-phosphate-specific receptors and is thought to occur at an

Lysosomal enzymes are subjected to a number of modifications including carbohydrate restructuring and proteolytic maturation. Some of these reactions support lysosomal targeting, others are necessary for activation or keeping the enzyme inactive before being segregated, while still others may be adventitious. The non-segregated fraction of the enzyme is secreted and can be isolated from the medium. It is considered that the secreted lysosomal enzymes fulfill certain physiological and pathophysiological roles. By comparing the secreted and the intracellular enzymes it is possible to distinguish between the reactions that occur before and after the segregation. In this review the reactions that may influence the segregation are referred to as the early processing and those characteristic for the enzymes isolated from lysosomal compartments as the late processing. The early processing is characterized mainly by modifications of carbohydrate side chains. In the late processing, proteolytic fragmentation represents the most conspicuous changes. The review focuses on the compartmentation of the reactions and the proteolytic fragmentation of lysosomal enzyme precursors. While a plethora of proteolytic reactions are involved, our knowledge of the proteinases responsible for the particular maturation reactions remains very limited. The review points also to work with cells from patients affected with lysosomal storage disorders, which contributed to our understanding of the lysosomal apparatus.