Function and properties of chimeric MPR 46-MPR 300 mannose 6-phosphate receptors.

The two known mannose 6-phosphate receptors (MPR 46 and MPR 300) mediate the transport of mannose 6-phosphate-containing lysosomal proteins to lysosomes. Endocytosis of extracellular mannose 6-phosphate ligands can only be mediated by MPR 300. Neither type of MPR appears to be sufficient for targetting the full complement of lysosomal enzymes to lysosomes. The complements of lysosomal enzymes transported by either of the two receptors are distinct but largely overlapping. Chimeric receptors were constructed in which the transmembrane and cytoplasmic domains of the two receptors were systematically exchanged. After expression of the chimeric receptors in cells lacking endogenous MPRs the binding of ligands, the subcellular distribution and the sorting efficiency for lysosomal enzymes were analyzed. All chimeras were functional, and their subcellular distribution was similar to that of wild type MPRs. The ability to endocytose lysosomal enzymes was restricted to receptors with the lumenal domain of MPR 300. The efficiency to sort lysosomal enzymes correlated with the lumenal and cytoplasmic domains of MPR 300. In contrast to the wild type receptors, a significant fraction of most of the chimeric receptors was misrouted to lysosomes, indicating that the signals determining the routing of MPRs have been fitted for the parent receptor polypeptides.

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.

Luciferase assembly after transport into mammalian microsomes involves molecular chaperones and peptidyl-prolyl cis/trans-isomerases.

The assembly of a heterodimeric luciferase was studied after de novo synthesis of corresponding precursor proteins in reticulocyte lysate and concomitant transport into dog pancreas microsomes. This cytosolic luciferase from a prokaryotic organism (Vibrio harveyi) was specifically used as a model protein to investigate (i) whether the eukaryotic cytosol and the microsomal lumen have similar folding capabilities and (ii) whether the requirements of a polypeptide for certain molecular chaperones and folding catalysts are determined by the polypeptide or the intracellular compartment. The two luciferase subunits were fused to the preprolactin signal peptide. Data indicate that efficient assembly of luciferase occurs in the mammalian microsomes. Furthermore, it was observed that luciferase assembly can be separated in time from synthesis and membrane transport, depends on ATP hydrolysis, is partially sensitive to cyclosporin A and FK506, and in the absence of lumenal proteins is less efficient as compared with the presence of lumenal proteins. Thus, heterodimeric luciferase depends on functionally related molecular chaperones and folding catalysts during its assembly in either the eukaryotic cytosol or the microsomal lumen.

Efficient folding of firefly luciferase after transport into mammalian microsomes in the absence of luminal chaperones and folding catalysts.

Folding of polypeptides emerging from the protein translocase in the membrane of mammalian microsomes was analyzed after synthesis of corresponding precursor proteins in a mammalian translation system. Firefly luciferase was used as a model protein; the corresponding hybrid precursor contained the preprolactin signal peptide. The rates and efficiencies of folding of luciferase in microsomes were compared with those of folding of luciferase in the cytosol. Furthermore, folding of luciferase in microsomes was compared with that in proteoliposomes, i.e. in the absence of luminal molecular chaperones and folding catalysts. Folding in microsomes was less efficient compared with folding in the cytosol. Folding in the absence of luminal proteins was more efficient compared with folding in their presence and identical to folding in the cytosol. Thus, firefly luciferase emerging from translocase can efficiently fold to its native conformation without chaperoning by any luminal proteins. There may be molecular chaperones present in the microsomal membrane that can efficiently substitute for the cytosolic chaperone machinery comprising Hsp40, Hsp60, and Hsp70 with respect to folding of firefly luciferase.

cDNA and derived amino acid sequence of the hypusine containing protein from Dictyostelium discoideum.

The eukaryotic translation initiation factor eIF-4D is the only protein known to contain the unusual amino acid hypusine, a posttranslationally modified lysine. For the production of monoclonal antibodies the hypusine-containing protein (HP) was isolated from Dictyostelium discoideum. Using these monoclonal antibodies, a full-length cDNA clone was isolated from a lambda gt11 library. The D. discoideum HP consists of 169 amino acids and has a molecular mass of 18.3 kDa. It is encoded by a single gene. Tryptic and cyanogen bromide peptides were prepared from the purified protein and sequenced. The hypusine residue is located at amino acid position 65 of the HP. The corresponding mRNA of approx. 0.6 kb is present throughout the life cycle of D. discoideum.