QIL1-dependent assembly of MICOS complex-lethal mutation in C19ORF70 resulting in liver disease and severe neurological retardation.

Seven subunits of the mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) in humans have been recently described in function and structure. QIL1 (also named MIC13) is a small complex that is crucial for the maintenance and assembling of MICOS. A novel mutation of an essential splice site in the C19orf70 gene encoding QIL1 induces severe mitochondrial encephalopathy, hepatopathy and lactate acidosis consistent with psychomotor retardation. In addition, bilateral kidney stones were observed. Disassembly of MICOS complex subunits displays lack of MIC10-MIC26-MIC27-QIL1 subcomplex, resulting in aberrant cristae structure and a loss of cristae junctions and contact sites. In liver and muscle tissue, the activity of the respiratory chain complexes (OXPHOS) was severely impaired. Defects in MICOS complex do not only affect mitochondrial architecture, but also mitochondrial fusion, metabolic signalling, lipid trafficking and cellular electric homeostasis.

[The frequency of older people living in nursing homes: results from a population-based study in Dortmund]. / Die Häufigkeit der Unterbringung älterer Menschen in stationären Pflegeeinrichtungen: Ergebnisse einer bevölkerungsbezogenen Analyse in der Stadt Dortmund.

Scientific studies often exclude institutionalized people. Thus, there is insufficient information about the percentage of older people, who are living in nursing homes. Furthermore, when they move to a care facility, it is questionable whether their new address is officially registered. This is a major prerequisite for their accessibility in studies. By using a standardized questionnaire, the number of nursing home residents in Dortmund was anonymously recorded. Their percentage of the population was determined separately for gender and age. This information was then compared to the official registry. Of those 65 years and older, 5.0% of women and 1.8% of men lived in long-term nursing homes. The percentage of institutionalized people of both genders increases with age; however, the correlation is stronger for women. Overall, 79.5% of the residents are female. To some extent, there were large differences between the information from the care facilities and the official registry concerning the number of residents.

The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein.

Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y.

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.

Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames.

As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins. Ynl080c-GFP and Ynl081c-GFP fluorescence was visible in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C-terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5'-GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP-Ynl020c and GFP-Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases.

Compact organization of rRNA genes in the filamentous fungus Ashbya gossypii.

The rDNA cluster in the phytopathogenic fungus Ashbya gossypii consists of approximately 50 tandem repeat units of 8197 bp. Each unit carries a gene for the 35S pre-rRNA, processed into 18S, 5.8S and 25S rRNA, and a divergently transcribed gene for 5S rRNA. The well-characterized rDNA of the yeast Saccharomyces cerevisiae is the only other example of a completely sequenced rDNA unit (9137 bp) carrying both a 35S pre-rRNA and a 5S rRNA gene. The coding regions for the 5S, 5.8S, 18S and 25S rRNAs are 95-100% identical whereas transcribed and non-transcribed spacers show 43-66% sequence identity. Functionally characterized rDNA and rRNA elements of S. cerevisiae can be unambiguously recognized in the A. gossypii sequence, including the RNA polymerase-I transcription start site, two Reb1p enhancer binding sites and numerous recognition sequences for rRNA modification and processing. In addition to these functionally characterized sequences eight highly conserved elements from 10 to 71 bp were detected in the over 600-bp transcribed region upstream of the 18S rRNA gene which most likely play as yet uncharacterized functions at the DNA or RNA level. In addition to this work we started to identify A. gossypii homologs of S. cerevisiae nucleolar proteins involved in rDNA maturation.

Alternative mechanisms for trafficking of lysosomal enzymes in mannose 6-phosphate receptor-deficient mice are cell type-specific.

Viable mice nullizygous in genes encoding the 300 kDa and the 46 kDa mannose 6-phosphate receptors (MPR 300 and MPR 46) and the insulin like growth factor II (IGF II) were generated to study the trafficking of lysosomal enzymes in the absence of MPRs. The mice have an I-cell disease-like phenotype, with increase of lysosomal enzymes in serum and normal activities in tissues. Surprisingly, the ability of MPR-deficient cells to transport newly synthesized lysosomal enzymes to lysosomes and the underlying mechanisms were found to depend on the cell type. MPR-deficient thymocytes target newly synthesized cathepsin D to lysosomes via an intracellular route. In contrast, hepatocytes and fibroblasts secrete newly synthesized cathepsin D. In fibroblasts recapture of secreted lysosomal enzymes, including that of cathepsin D, is limited and results in lysosomal storage, both in vivo and in vitro, whereas recapture by hepatocytes is remarkably effective in vivo and can result in lysosomal enzyme levels even above normal.

I-cell disease-like phenotype in mice deficient in mannose 6-phosphate receptors.

Mannose 6-phosphate receptor deficient mice were generated by crossing mice carrying null alleles for Igf2 and the 300 kDa and 46 kDa mannose 6-phosphate receptors, Mpr300 and Mpr46. Pre- and perinatal lethality of mice nullizygous for Igf2, Mpr300 and Mpr46 was increased. Triple deficient mice surviving the first postnatal day had normal viability and developed a phenotype resembling human I-cell disease. The triple deficient mice were characterized by dwarfism, facial dysplasia, waddling gait, dysostosis multiplex, elevated lysosomal enzymes in serum and histological signs of lysosomal storage predominantly in fibroblasts, but also in parenchymal cells of brain and liver. A paternally inherited Mpr300 wild type allele that is normally inactive in mice due to imprinting was reactivated in some tissues of mice lacking IGF II and MPR 46 and carrying a maternal Mpr300 null allele. Inspite of the partial reactivation the phenotype of these mice was similar to that of triple deficient mice.

Identification of three internalization sequences in the cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor.

The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

Serine phosphorylation site of the 46-kDa mannose 6-phosphate receptor is required for transport to the plasma membrane in Madin-Darby canine kidney and mouse fibroblast cells.

Up to 4% of the human 46-kDa mannose 6-phosphate receptor (MPR46) expressed in Madin-Darby canine kidney (MDCK) cells are localized at the cell surface. At steady state, the expression of MPR46 on the apical surface of filter-grown MDCK cells is about sixfold lower than on the basolateral surface. The cytoplasmic domain of the MPR46 is phosphorylated on serine 56 at low stoichiometry. By expressing mutant MPR46 we have shown that the MPR46 phosphorylation site is required for delivery to the plasma membrane. In addition, mutant MPR46 expressed in MPR-deficient mouse embryonic fibroblasts were not detected at the cell surface and their ability to sort newly synthesized cathepsin D was not altered. Since the loss of MPR46 phosphorylation correlates with the lack of cell surface expression, phosphorylation of serine 56 may either function as a direct plasma membrane targeting signal or inhibit MPR46 recycling from endosomes to Golgi, resulting in trafficking to the cell surface.

The phosphorylation pattern of oligosaccharides in secreted procathepsin D is glycosylation site-specific and independent of the expression of mannose 6-phosphate receptors.

Mammalian cells contain two types of mannose 6-phosphate receptors (MPR), MPRs 46 and 300, that contribute with variable efficiency to the sorting of individual lysosomal proteins. To evaluate the role of phosphorylated oligosaccharides for the sorting efficiency by either of the two receptors, the structure of phosphorylated oligosaccharides on lysosomal proteins escaping sorting in cells lacking MPR 46 and/or MPR 300 was analyzed. Procathepsin D was chosen as a model because it is sorted efficiently via MPR 300 and poorly via MPR 46 and contains a distinct and highly heterogenous mixture of phosphorylated oligosaccharides at either of its two N-glycosylation sites. Both MPRs 46 and 300 were found to have a minor but distinct preference for forms of procathepsin D and other lysosomal proteins containing oligosaccharides with two phosphomonoesters. However, the phosphorylation of oligosaccharides in procathepsin D and other lysosomal proteins that escape sorting in control cells or in cells lacking MPR 46 and/or MPR 300 was strikingly similar, and oligosaccharides with two phosphomonoesters represented the major oligosaccharide species. We conclude from these results that the position of the position of the phosphate groups, the structure of the underlying oligosaccharide, and/or the polypeptide backbone of lysosomal proteins have major roles in determining the affinity to MPRs.

The 46 kDa mannose-6-phosphate receptor contains a signal for basolateral sorting within the 19 juxtamembrane cytosolic residues.

The cytosolic domain of the 46 kDa mannose-6-phosphate receptor (MPR 46) contains a signal that mediates sorting of the receptor and of a reporter protein to the basolateral surface domain of Madin-Darby canine kidney cells. Progressive truncation of the 67 cytosolic residues indicated that the 19 juxtamembrane residues are sufficient for basolateral sorting. Alanine/glycine-scanning mutagenesis identified Glu-11 and Ala-17 as the critical residues between residues 7 and 19. Glu-11 is also of critical importance for the one of the three internalization signals in the cytosolic tail of the receptor [Denzer, Weber, Hille-Rehfeld, von Figura and Pohlmann (1997) Biochem. J. 326, 497-505]. Although overlapping, the signals for basolateral sorting and internalization depend on different residues. The basolateral sorting signal of MPR 46 is distinct from tyrosine- or dileucine-based basolateral sorting signals and also lacks similarity to the few other basolateral signals that do not fall into these two classes.

Expression of mannose 6-phosphate receptors in chicken.

In mammals, the sorting of newly synthesized lysosomal enzymes is accomplished by two mannose 6-phosphate receptors (MPR) designated MPR46 and MPR300. MPR300 has an additional function in clearing the nonglycosylated insulin-like growth factor II (IGFII). The distinct expression pattern of the two MPR has been ascribed to the control of MPR300 expression by IGFII. In lower vertebrates, such as chickens or frogs, only MPR300 homologues have been described. These MPR300 homologues do not bind IGFII. In the present study, we examined whether lower vertebrates such as chickens also express two types of MPR and, if so, whether the expression pattern is distinct or similar. We were able to clone chicken cDNA fragments homologous to mammalian MPR46 and MPR300 and to show the synthesis of respective MPR polypeptides, thus establishing the existence of two types of MPR also in a nonmammalian species. Further, we analyzed the expression of the two MPR in chicken by Northern blotting and in situ hybridization. High levels of MPR46 and MPR300 RNA were detectable in epithelia, ganglia, and uropoietic system of chicken embryos. In a number of embryonic and adult tissues, varying ratios of MPR46 and MPR300 RNA were observed. The expression pattern for both MPR46 and MPR300 was distinct, although less pronounced than in mice. We conclude that functional differences unrelated to the additional function of the mammalian MPR300 as a receptor clearing IGFII are responsible for the distinct expression of the two MPR in nonmammalian, and probably also in mammalian, species.

Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes.

Mouse embryonic fibroblasts that are deficient in the two mannose 6-phosphate receptors (MPRs) MPR 46 and MPR 300 missort the majority (> or = 85%) of soluble lysosomal proteins into the medium. Human MPR 46 and MPR 300 were expressed in these cells to test whether overexpression of a single type of MPR can restore transport of lysosomal proteins to lysosomes. Only a partial correction of the missorting was observed after overexpression of MPR 46. Even at MPR 46 levels that are five times higher than the wild-type level, more than one third of the newly synthesized lysosomal proteins accumulates in the secretions. Two-fold overexpression of MPR 300 completely corrects the missorting of lysosomal enzymes. However, at least one fourth of the lysosomal enzymes are transported along a secretion-recapture pathway that is sensitive to mannose 6-phosphate in medium. In control fibroblasts that express both types of MPR, the secretion-recapture pathway is of minor importance. These results imply that neither overexpression of MPR 46 nor MPR 300 is sufficient for targeting of lysosomal proteins along intracellular routes.

Sequencing a cosmid clone of Saccharomyces cerevisiae chromosome XIV reveals 12 new open reading frames (ORFs) and an ancient duplication of six ORFs.

A sequence of 31431 bp located on the left arm of chromosome (chr.) XIV from Saccharomyces cerevisiae was analysed. A total of 18 open reading frames (ORFs) could be identified. Twelve ORFs are new, two of which are most likely ribosomal protein genes, leaving ten ORFs of unknown function. Nine of the 18 ORFs show either at least 20% overall amino acid identity or significant regional homology to other S. cerevisiae ORFs. Additionally, six of these nine ORFs have homologues of similar size and the same transcriptional orientation within a stretch of 50 kb on chromosome IX. The degree of homology ranges from 90% overall identity to 23% in 375 amino acids. The homologues on chromosome IX are grouped in two blocks that are separated by relatively long ORFs. This is the first example of a multi-gene duplication in S. cerevisiae not linked to a centromere or subtelomere region.

The two mannose 6-phosphate receptors transport distinct complements of lysosomal proteins.

Mammalian cells express two different mannose 6-phosphate receptors (MPR 46 and MPR 300), which both mediate targeting of Man-6-P-containing lysosomal proteins to lysosomes. To assess the contribution of either and both MPRs to the transport of lysosomal proteins, fibroblasts were established from mouse embryos that were homozygous for disrupted alleles of either MPR 46 or MPR 300 or both MPRs. Fibroblasts missing both MPRs secreted most of the newly synthesized lysosomal proteins and were unable to maintain the catabolic function of lysosomes. The intracellular levels of lysosomal proteins decreased to < 20%, and undigested material accumulated in the lysosomal compartment. Fibroblasts lacking either MPR exhibited only a partial missorting and maintained, in general, half-normal to normal levels of lysosomal proteins. The same species of lysosomal proteins were found in secretions of double MPR-deficient fibroblasts as in secretions of single MPR-deficient fibroblasts, but at different ratios. This clearly indicates that neither MPR has an exclusive affinity for one or several lysosomal proteins. Furthermore, neither MPR can substitute in vivo for the loss of the other. It is proposed that the heterogeneity of the Man-6-P recognition marker within a lysosomal protein and among different lysosomal proteins has necessitated the evolution of two MPRs with complementary binding properties to ensure an efficient targeting of lysosomal proteins.

New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae.

We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.

Mistargeting of lysosomal enzymes in M(r) 46,000 mannose 6-phosphate receptor-deficient mice is compensated by carbohydrate-specific endocytotic receptors.

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor (MPR 46) in mice is associated with normal levels of lysosomal enzymes in the circulation, while in MPR 46-deficient cells an increased secretion of lysosomal enzymes is apparent [Köster, A., Saftig, P., Matzner, U., von Figura, K., Peters, C. & Pohlmann, R. (1993) EMBO J. 12, 5219-5223]. This points to the existence of mechanisms that prevent or compensate for mistargeting of lysosomal enzymes in vivo. In the present study, we have injected inhibitors of three carbohydrate-specific endocytotic receptors into MPR 46-deficient and control mice. Inhibition of these receptors was associated with a pronounced increase of three lysosomal enzymes in the serum of MPR 46-deficient mice. These results clearly show that lysosomal enzymes are mistargeted in MPR 46-deficient mice and that carbohydrate-specific endocytotic receptors are part of the mechanisms that compensate for the mistargeting of lysosomal enzymes in MPR 46-deficient mice. Moreover, evidence was obtained that, also in control mice, the steady-state level of some lysosomal enzyme is controlled by these receptors.

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.