Synthesis of the cation-dependent mannose 6-phosphate-specific receptor was followed in cells of human (fibroblasts, Hep G2 cells, U937 monocytes, blood-derived macrophages) or rat (Morris hepatoma 7777 cells), origin. The mature form of the receptor has an apparent molecular size of 46 kDa except in fibroblasts, where the apparent molecular size was 43 kDa. The receptor contains 7-8 N-linked oligosaccharide chains, about 5 of which are converted into endo -resistant forms within 2 h of synthesis. A small fraction of the receptor (about 3% of total in U937 monocytes) is located at the cell surface while the bulk of the receptor resides in internal membranes. Part of the internal receptors (20% in fibroblasts) resides in membranes of the endocy tic pathway. The receptor was not detectable in dense lysosomes. The receptor is a hydrophobic transmembrane protein partitioning with Triton X-l 14. The cy tosolic portion of the receptor comprises a molecular size of about 5 kDa and contains the C-terminus. The luminal (or external) portion of the receptor comprises a molecular size of > 37.5 kDa, of which more than half is represented by carbohydrate. Cross-linking experiments suggest that the mature receptor exists in membranes as a dimer. 46-kDa-Mannose-6-phosphat-spezifischer Rezeptor: Biosynthese, Processing, subzelluläre Lokalisation und Topologie Zusammenfassung: In verschiedenen menschlichen Zellinien (Fibroblasten, Hep G2-Zellen, U937 Monocyten, Makrophagen) und MorrisHepatoma 7777-Zellen der Ratte wurde die Biosynthese des Kationen-abhängigen Mannose-6-phosphat-spezifischen Rezeptors untersucht. Der reife Rezeptor hat in Fibroblasten eine Molekularmasse von 43 kDa und in den übrigen Zellen eine Molekularmasse von 46 kDa. Der Rezeptor enthält 7-8 Asparagin-verknüpfte Oligosaccharide, von denen ca. 5 innerhalb von 2 h nach der Synthese in Endo-H-resistente Formen umgewandelt werden. Der Rezeptor ist größtenteils in intrazellulären Membranen lokalisiert. Ein Teil der intrazellulären Rezeptoren (20% in Fibroblasten) ist in Membranen des

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70‐fold higher enzyme activities of acid phosphatase than non‐transfected BHK cells. The CT29‐LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans‐Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29‐LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose‐6‐phosphate residues suggested that transport does not involve mannose‐6‐phosphate receptors. Part of the membrane‐associated CT29‐LAP was processed to a soluble form. The mechanism that converts CT29‐LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29‐derived cRNA in the presence of microsomal membranes yielded a CT29‐LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29‐LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29‐LAP is released from the membrane by proteolytic cleavage, which removes a C‐terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.

Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in Xgtll. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids (Mr = 30,993), and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential Nglycosylation sites from a C-terminal region lacking Nglycosylation sites. Thus the N-terminal (Mr = 18,299) and C-terminal (Mr ' 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12.

A 2112‐bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3‐kb mRNA from human liver and HL‐60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12‐bp 5′ non‐coding region, an open reading frame of 1269 bp and an 831‐bp 3′ non‐coding region with a putative polyadenylation signal 25 bp upstream of a 3′ poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N‐terminal amino acid residues of human prostatic acid phosphatase and the N‐terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.

The nucleotide sequences of cloned cDNAs encoding the precursors for vasotocin and isotocin have been elucidated by analyzing a Agtll library constructed from poly(A)' RNA from the hypothalamic region of the teleost fish Catostomus commersom'. Screening of the library was carried out with synthetic oligonucleotide probes deduced from the amino acid sequences of the nonapeptides vasotocin and isotocin. The cDNA nucleotide sequences predict isotocin and vasotocin prohormone precursors each consisting of a signal peptide, a hormone moiety, and a neurophysin-like molecule. However, in comparison to their mammalian counterparts, both fish neurophysins are extended at their C termini by an =30 amino acid sequence with a leucine-rich core segment. These extensions show striking similarities with the glycopeptide moiety (the so-called copeptin) present in mammalian vasopressin precursors, except that they lack the consensus sequence for N-glycosylation. These data suggest that mammalian copeptin is derived from the C terminus of an ancestral neurophysin.