Fungal Planet description sheets: 951-1041.

Novel species of fungi described in this study include those from various countries as follows: Antarctica, Apenidiella antarctica from permafrost, Cladosporium fildesense from an unidentified marine sponge. Argentina, Geastrum wrightii on humus in mixed forest. Australia, Golovinomyces glandulariae on Glandularia aristigera, Neoanungitea eucalyptorum on leaves of Eucalyptus grandis, Teratosphaeria corymbiicola on leaves of Corymbia ficifolia, Xylaria eucalypti on leaves of Eucalyptus radiata. Brazil, Bovista psammophila on soil, Fusarium awaxy on rotten stalks of Zea mays, Geastrum lanuginosum on leaf litter covered soil, Hermetothecium mikaniae-micranthae (incl. Hermetothecium gen. nov.) on Mikania micrantha, Penicillium reconvexovelosoi in soil, Stagonosporopsis vannaccii from pod of Glycine max. British Virgin Isles, Lactifluus guanensis on soil. Canada, Sorocybe oblongispora on resin of Picea rubens. Chile, Colletotrichum roseum on leaves of Lapageria rosea. China, Setophoma caverna from carbonatite in Karst cave. Colombia, Lareunionomyces eucalypticola on leaves of Eucalyptus grandis. Costa Rica, Psathyrella pivae on wood. Cyprus, Clavulina iris on calcareous substrate. France, Chromosera ambigua and Clavulina iris var. occidentalis on soil. French West Indies, Helminthosphaeria hispidissima on dead wood. Guatemala, Talaromyces guatemalensis in soil. Malaysia, Neotracylla pini (incl. Tracyllales ord. nov. and Neotracylla gen. nov.) and Vermiculariopsiella pini on needles of Pinus tecunumanii. New Zealand, Neoconiothyrium viticola on stems of Vitis vinifera, Parafenestella pittospori on Pittosporum tenuifolium, Pilidium novae-zelandiae on Phoenix sp. Pakistan, Russula quercus-floribundae on forest floor. Portugal, Trichoderma aestuarinum from saline water. Russia, Pluteus liliputianus on fallen branch of deciduous tree, Pluteus spurius on decaying deciduous wood or soil. South Africa, Alloconiothyrium encephalarti, Phyllosticta encephalarticola and Neothyrostroma encephalarti (incl. Neothyrostroma gen. nov.) on leaves of Encephalartos sp., Chalara eucalypticola on leaf spots of Eucalyptus grandis × urophylla, Clypeosphaeria oleae on leaves of Olea capensis, Cylindrocladiella postalofficium on leaf litter of Sideroxylon inerme, Cylindromonium eugeniicola (incl. Cylindromonium gen. nov.) on leaf litter of Eugenia capensis, Cyphellophora goniomatis on leaves of Gonioma kamassi, Nothodactylaria nephrolepidis (incl. Nothodactylaria gen. nov. and Nothodactylariaceae fam. nov.) on leaves of Nephrolepis exaltata, Falcocladium eucalypti and Gyrothrix eucalypti on leaves of Eucalyptus sp., Gyrothrix oleae on leaves of Olea capensis subsp. macrocarpa, Harzia metrosideri on leaf litter of Metrosideros sp., Hippopotamyces phragmitis (incl. Hippopotamyces gen. nov.) on leaves of Phragmites australis, Lectera philenopterae on Philenoptera violacea, Leptosillia mayteni on leaves of Maytenus heterophylla, Lithohypha aloicola and Neoplatysporoides aloes on leaves of Aloe sp., Millesimomyces rhoicissi (incl. Millesimomyces gen. nov.) on leaves of Rhoicissus digitata, Neodevriesia strelitziicola on leaf litter of Strelitzia nicolai, Neokirramyces syzygii (incl. Neokirramyces gen. nov.) on leaf spots of Syzygium sp., Nothoramichloridium perseae (incl. Nothoramichloridium gen. nov. and Anungitiomycetaceae fam. nov.) on leaves of Persea americana, Paramycosphaerella watsoniae on leaf spots of Watsonia sp., Penicillium cuddlyae from dog food, Podocarpomyces knysnanus (incl. Podocarpomyces gen. nov.) on leaves of Podocarpus falcatus, Pseudocercospora heteropyxidicola on leaf spots of Heteropyxis natalensis, Pseudopenidiella podocarpi, Scolecobasidium podocarpi and Ceramothyrium podocarpicola on leaves of Podocarpus latifolius, Scolecobasidium blechni on leaves of Blechnum capense, Stomiopeltis syzygii on leaves of Syzygium chordatum, Strelitziomyces knysnanus (incl. Strelitziomyces gen. nov.) on leaves of Strelitzia alba, Talaromyces clemensii from rotting wood in goldmine, Verrucocladosporium visseri on Carpobrotus edulis. Spain, Boletopsis mediterraneensis on soil, Calycina cortegadensisi on a living twig of Castanea sativa, Emmonsiellopsis tuberculata in fluvial sediments, Mollisia cortegadensis on dead attached twig of Quercus robur, Psathyrella ovispora on soil, Pseudobeltrania lauri on leaf litter of Laurus azorica, Terfezia dunensis in soil, Tuber lucentum in soil, Venturia submersa on submerged plant debris. Thailand, Cordyceps jakajanicola on cicada nymph, Cordyceps kuiburiensis on spider, Distoseptispora caricis on leaves of Carex sp., Ophiocordyceps khonkaenensis on cicada nymph. USA, Cytosporella juncicola and Davidiellomyces juncicola on culms of Juncus effusus, Monochaetia massachusettsianum from air sample, Neohelicomyces melaleucae and Periconia neobrittanica on leaves of Melaleuca styphelioides × lanceolata, Pseudocamarosporium eucalypti on leaves of Eucalyptus sp., Pseudogymnoascus lindneri from sediment in a mine, Pseudogymnoascus turneri from sediment in a railroad tunnel, Pulchroboletus sclerotiorum on soil, Zygosporium pseudomasonii on leaf of Serenoa repens. Vietnam, Boletus candidissimus and Veloporphyrellus vulpinus on soil. Morphological and culture characteristics are supported by DNA barcodes.

Fungal Planet description sheets: 716-784.

Novel species of fungi described in this study include those from various countries as follows: Australia, Chaetopsina eucalypti on Eucalyptus leaf litter, Colletotrichum cobbittiense from Cordyline stricta × C. australis hybrid, Cyanodermella banksiae on Banksia ericifolia subsp. macrantha, Discosia macrozamiae on Macrozamia miquelii, Elsinoë banksiigena on Banksia marginata, Elsinoë elaeocarpi on Elaeocarpus sp., Elsinoë leucopogonis on Leucopogon sp., Helminthosporium livistonae on Livistona australis, Idriellomyces eucalypti (incl. Idriellomyces gen. nov.) on Eucalyptus obliqua, Lareunionomyces eucalypti on Eucalyptus sp., Myrotheciomyces corymbiae (incl. Myrotheciomyces gen. nov., Myrotheciomycetaceae fam. nov.), Neolauriomyces eucalypti (incl. Neolauriomyces gen. nov., Neolauriomycetaceae fam. nov.) on Eucalyptus sp., Nullicamyces eucalypti (incl. Nullicamyces gen. nov.) on Eucalyptus leaf litter, Oidiodendron eucalypti on Eucalyptus maidenii, Paracladophialophora cyperacearum (incl. Paracladophialophoraceae fam. nov.) and Periconia cyperacearum on leaves of Cyperaceae, Porodiplodia livistonae (incl. Porodiplodia gen. nov., Porodiplodiaceae fam. nov.) on Livistona australis, Sporidesmium melaleucae (incl. Sporidesmiales ord. nov.) on Melaleuca sp., Teratosphaeria sieberi on Eucalyptus sieberi, Thecaphora australiensis in capsules of a variant of Oxalis exilis. Brazil, Aspergillus serratalhadensis from soil, Diaporthe pseudoinconspicua from Poincianella pyramidalis, Fomitiporella pertenuis on dead wood, Geastrum magnosporum on soil, Marquesius aquaticus (incl. Marquesius gen. nov.) from submerged decaying twig and leaves of unidentified plant, Mastigosporella pigmentata from leaves of Qualea parviflorae, Mucor souzae from soil, Mycocalia aquaphila on decaying wood from tidal detritus, Preussia citrullina as endophyte from leaves of Citrullus lanatus, Queiroziella brasiliensis (incl. Queiroziella gen. nov.) as epiphytic yeast on leaves of Portea leptantha, Quixadomyces cearensis (incl. Quixadomyces gen. nov.) on decaying bark, Xylophallus clavatus on rotten wood. Canada, Didymella cari on Carum carvi and Coriandrum sativum. Chile, Araucasphaeria foliorum (incl. Araucasphaeria gen. nov.) on Araucaria araucana, Aspergillus tumidus from soil, Lomentospora valparaisensis from soil. Colombia, Corynespora pseudocassiicola on Byrsonima sp., Eucalyptostroma eucalyptorum on Eucalyptus pellita, Neometulocladosporiella eucalypti (incl. Neometulocladosporiella gen. nov.) on Eucalyptus grandis × urophylla, Tracylla eucalypti (incl. Tracyllaceae fam. nov., Tracyllalales ord. nov.) on Eucalyptus urophylla. Cyprus, Gyromitra anthracobia (incl. Gyromitra subg. Pseudoverpa) on burned soil. Czech Republic, Lecanicillium restrictum from the surface of the wooden barrel, Lecanicillium testudineum from scales of Trachemys scripta elegans. Ecuador, Entoloma yanacolor and Saproamanita quitensis on soil. France, Lentithecium carbonneanum from submerged decorticated Populus branch. Hungary, Pleuromyces hungaricus (incl. Pleuromyces gen. nov.) from a large Fagus sylvatica log. Iran, Zymoseptoria crescenta on Aegilops triuncialis. Malaysia, Ochroconis musicola on Musa sp. Mexico, Cladosporium michoacanense from soil. New Zealand , Acrodontium metrosideri on Metrosideros excelsa, Polynema podocarpi on Podocarpus totara, Pseudoarthrographis phlogis (incl. Pseudoarthrographis gen. nov.) on Phlox subulata. Nigeria, Coprinopsis afrocinerea on soil. Pakistan, Russula mansehraensis on soil under Pinus roxburghii. Russia, Baorangia alexandri on soil in deciduous forests with Quercus mongolica. South Africa, Didymocyrtis brachylaenae on Brachylaena discolor. Spain, Alfaria dactylis from fruit of Phoenix dactylifera, Dothiora infuscans from a blackened wall, Exophiala nidicola from the nest of an unidentified bird, Matsushimaea monilioides from soil, Terfezia morenoi on soil. United Arab Emirates, Tirmania honrubiae on soil. USA, Arxotrichum wyomingense (incl. Arxotrichum gen. nov.) from soil, Hongkongmyces snookiorum from submerged detritus from a fresh water fen, Leratiomyces tesquorum from soil, Talaromyces tabacinus on leaves of Nicotiana tabacum. Vietnam, Afroboletus vietnamensis on soil in an evergreen tropical forest, Colletotrichum condaoense from Ipomoea pes-caprae. Morphological and culture characteristics along with DNA barcodes are provided.

Fungal Planet description sheets: 558-624.

Novel species of fungi described in this study include those from various countries as follows: Australia: Banksiophoma australiensis (incl. Banksiophoma gen. nov.) on Banksia coccinea, Davidiellomycesaustraliensis (incl. Davidiellomyces gen. nov.) on Cyperaceae, Didymocyrtis banksiae on Banksia sessilis var. cygnorum, Disculoides calophyllae on Corymbia calophylla, Harknessia banksiae on Banksia sessilis, Harknessia banksiae-repens on Banksia repens, Harknessia banksiigena on Banksia sessilis var. cygnorum, Harknessia communis on Podocarpus sp., Harknessia platyphyllae on Eucalyptus platyphylla, Myrtacremonium eucalypti (incl. Myrtacremonium gen. nov.) on Eucalyptus globulus, Myrtapenidiella balenae on Eucalyptus sp., Myrtapenidiella eucalyptigena on Eucalyptus sp., Myrtapenidiella pleurocarpae on Eucalyptuspleurocarpa, Paraconiothyrium hakeae on Hakea sp., Paraphaeosphaeria xanthorrhoeae on Xanthorrhoea sp., Parateratosphaeria stirlingiae on Stirlingia sp., Perthomyces podocarpi (incl. Perthomyces gen. nov.) on Podocarpus sp., Readeriella ellipsoidea on Eucalyptus sp., Rosellinia australiensis on Banksia grandis, Tiarosporella corymbiae on Corymbia calophylla, Verrucoconiothyriumeucalyptigenum on Eucalyptus sp., Zasmidium commune on Xanthorrhoea sp., and Zasmidium podocarpi on Podocarpus sp. Brazil: Cyathus aurantogriseocarpus on decaying wood, Perenniporia brasiliensis on decayed wood, Perenniporia paraguyanensis on decayed wood, and Pseudocercospora leandrae-fragilis on Leandrafragilis.Chile: Phialocephala cladophialophoroides on human toe nail. Costa Rica: Psathyrella striatoannulata from soil. Czech Republic: Myotisia cremea (incl. Myotisia gen. nov.) on bat droppings. Ecuador: Humidicutis dictiocephala from soil, Hygrocybe macrosiparia from soil, Hygrocybe sangayensis from soil, and Polycephalomyces onorei on stem of Etlingera sp. France: Westerdykella centenaria from soil. Hungary: Tuber magentipunctatum from soil. India: Ganoderma mizoramense on decaying wood, Hodophilus indicus from soil, Keratinophyton turgidum in soil, and Russula arunii on Pterigota alata.Italy: Rhodocybe matesina from soil. Malaysia: Apoharknessia eucalyptorum, Harknessia malayensis, Harknessia pellitae, and Peyronellaea eucalypti on Eucalyptus pellita, Lectera capsici on Capsicum annuum, and Wallrothiella gmelinae on Gmelina arborea.Morocco: Neocordana musigena on Musa sp. New Zealand: Candida rongomai-pounamu on agaric mushroom surface, Candida vespimorsuum on cup fungus surface, Cylindrocladiella vitis on Vitis vinifera, Foliocryphia eucalyptorum on Eucalyptus sp., Ramularia vacciniicola on Vaccinium sp., and Rhodotorula ngohengohe on bird feather surface. Poland: Tolypocladium fumosum on a caterpillar case of unidentified Lepidoptera.Russia: Pholiotina longistipitata among moss. Spain: Coprinopsis pseudomarcescibilis from soil, Eremiomyces innocentii from soil, Gyroporus pseudocyanescens in humus, Inocybe parvicystis in humus, and Penicillium parvofructum from soil. Unknown origin: Paraphoma rhaphiolepidis on Rhaphiolepsis indica.USA: Acidiella americana from wall of a cooling tower, Neodactylaria obpyriformis (incl. Neodactylaria gen. nov.) from human bronchoalveolar lavage, and Saksenaea loutrophoriformis from human eye. Vietnam: Phytophthora mekongensis from Citrus grandis, and Phytophthora prodigiosa from Citrus grandis. Morphological and culture characteristics along with DNA barcodes are provided.

Use of light and scanning electron microscopy to examine colonisation of barley rhizosphere by the nematophagous fungus Verticillium chlamydosporium.

Barley roots were readily colonised by the nematophagous fungus Verticillium chlamydosporium. Light microscopy (LM) but also low temperature scanning electron microscopy (LTSEM) revealed details of the colonisation process. Hyphae were found on the rhizoplane often with dictyochlamydospores. Hyphae of V. chlamydosporium penetrated epidermal cells, often by means of appressoria. A hyphal network was formed in epidermal and cortical cells. Likewise, hyphal coils were found within root cells next to transverse cell walls. Cortical cells were the limits of fungal colonisation, since no hyphae were seen in the vascular cylinder. Modifications of root cell contents (phenolic droplets and callose appositions) were common three weeks after inoculation with V. chlamydosporium. These features may indicate induction of plant defence reactions in late stages of root colonisation by the fungus. Both LTSEM and LM have proved extremely useful to describe root colonisation by the fungus. The results found may have implications in the mode action of nematophagous fungi against plant parasitic nematodes.

Colonization of plant roots by egg-parasitic and nematode-trapping fungi.

• The ability of the nematode-trapping fungus Arthrobotrys oligospora and the nematode egg parasite Verticillium chlamydosporium to colonize barley (Hordeum vulgare) and tomato (Lycopersicum esculentum) roots was examined, together with capability of the fungi to induce cell wall modifications in root cells. • Chemotropism was studied using an agar plate technique. Root colonization was investigated with light microscopy and scanning electron microscopy, while compounds involved in fungus-plant interactions were studied histochemically. • Only A. oligospora responded chemotropically to roots. Colonization of barley and tomato by both fungi involved appressoria to facilitate epidermis penetration. V. chlamydosporium colonized tomato root epidermis and produced chlamydospores. Papillae, appositions and lignitubers ensheathing hyphae on tomato were also found. Phenolics (including lignin), protein deposits and callose were present in papillae in both hosts. Both fungi were still present in epidermal cells 3 months after inoculation. • Nematophagous fungi colonized endophytically monocotyledon and dicotyledon plant roots. Arthrobotrys oligospora seemed to be more aggressive than V. chlamydosporium on barley roots. Both fungi induced cell wall modifications, but these did not prevent growth. The response of root cells to colonization by nematophagous fungi may have profound implications in the performance of these organisms as biocontrol agents of plant parasitic nematodes.

Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation.

Remodeling of fibrillar collagen in mouse tissues has been widely attributed to the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the main collagenase identified in this species. This proposal has been largely based on the repeatedly unproductive attempts to detect the presence in murine tissues of interstitial collagenase (MMP-1), a major collagenase in many species, including humans. In this work, we have performed an extensive screening of murine genomic and cDNA libraries using as probe the full-length cDNA for human MMP-1. We report the identification of two novel members of the MMP gene family which are contained within the cluster of MMP genes located at murine chromosome 9. The isolated cDNAs contain open reading frames of 464 and 463 amino acids and are 82% identical, displaying all structural features characteristic of archetypal MMPs. Comparison for sequence similarities revealed that the highest percentage of identities was found with human interstitial collagenase (MMP-1). The new proteins were tentatively called Mcol-A and Mcol-B (Murine collagenase-like A and B). Analysis of the enzymatic activity of the recombinant proteins revealed that both are catalytically autoactivable but only Mcol-A is able to degrade synthetic peptides and type I and II fibrillar collagen. Both Mcol-A and Mcol-B genes are located in the A1-A2 region of mouse chromosome 9, Mcol-A occupying a position syntenic to the human MMP-1 locus at 11q22. Analysis of the expression of these novel MMPs in murine tissues revealed their predominant presence during mouse embryogenesis, particularly in mouse trophoblast giant cells. According to their structural and functional characteristics, we propose that at least one of these novel members of the MMP family, Mcol-A, may play roles as interstitial collagenase in murine tissues and could represent a true orthologue of human MMP-1.

Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation.

The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system. This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well. As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery. Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon. In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded. This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system. When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in Delta(hrd3) cells. Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules.

A RING-H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae.

Der3/Hrd1p is a protein required for proper degradation of misfolded soluble and integral membrane proteins in the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. It is located to the ER membrane and consists of a N-terminal hydrophobic region with several transmembrane domains and a large hydrophilic tail oriented to the ER lumen containing a RING finger motif of the H2 class. We had previously reported that a truncated version of Der3p, Der3deltaRp, lacking 111 residues of the lumenal domain including the RING finger motif is not functional, suggesting the involvement of this domain in the function of the protein in ER degradation. We substantiated this hypothesis by constructing a mutated form of Der3/Hrd1p replacing the last cysteine of the motif with a serine. This mutated Der3(C399S) protein maintains the correct localization and topology of the wild-type protein, however, is not able to support the degradation of soluble and integral membrane proteins. This point mutation altering the RING-H2 motif behaves as a dominant allele especially when overexpressed from a 2mu plasmid by this increasing the half-life of CPY* more than 6-fold when compared with a wild-type strain. Furthermore coexpression of der3(C399S) with the wild-type allele is also able to partially suppress the temperature sensitive growth phenotype of a sec61-2 strain. Finally we have shown that overexpression of Hrd3p suppresses the dominant effect of the der3(C399S) mutation. These results could be explained by a competition between wild-type and mutant Der3 protein for the interaction with some other component of the ER degradation pathway, probably Hrd3p.

Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins.

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3-1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61-2 allele. This is accompanied by the stabilization of the Sec61-2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61-2 strain at the permissive temperature of 25 degrees C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61-2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.

Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation.

Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly folded proteins to their site of action and contains a quality-control system to monitor and prevent abnormal proteins from being delivered. Many of these proteins are degraded by the cytoplasmic proteasome, which requires their retrograde transport to the cytoplasm. Based on a co-immunoprecipitation of major histocompatibility complex (MHC) class I heavy-chain breakdown intermediates with the translocon subunit Sec61p, it was speculated that Sec61p maybe involved in retrograde transport. Here we present functional evidence from genetic studies that Sec61p mediates retrograde transport of a mutated lumenal yeast carboxypeptidase ycsY (CPY*) in vivo. The endoplasmic reticulum lumenal chaperone BiP (Kar2p) and Sec63p, which are also subunits of the import machinery, are involved in export of CPY* to the cytosol. Thus our results demonstrate that retrograde transport of proteins is mediated by a functional translocon. We consider the export of endoplasmic reticulum-localized proteins to the cytosol by the translocon for proteasome degradation to be a general process in eukaryotic cell biology.

Identification of regulatory proteins that might be involved in carbon catabolite repression of the aminopeptidase I gene of the yeast Saccharomyces cerevisiae.

Transcription of the vacuolar aminopeptidase yscI (APE1) gene in Saccharomyces cerevisiae has previously been suggested to require the participation of a cis upstream activation sequence (UAS) involved in carbon catabolite repression that responds to glucose. To determine the structure of the APE1 UAS element, we used the 18-bp sequence 5'-ATGAATTAGTCAGCTTCT-3' as the DNA-binding site. Using gel mobility shift assays, we have identified a 78 kDa protein from yeast that binds specifically to both single and double-stranded forms of the UAS DNA-binding site. We have also identified a 48 kDa heterodimer from yeast that binds specifically to the single-stranded form of the UAS and whose DNA binding activity is remarkably heat stable. Even though the APE1 UAS contains a consensus sequence for the binding of the yeast activator protein yAP1, the two DNA-protein complexes could still be detected in a strain bearing a deletion in the YAP1 gene.

Transcriptional regulation of the yeast vacuolar aminopeptidase yscI encoding gene (APE1) by carbon sources.

Transcription of the vacuolar aminopeptidase yscI-encoding gene (APE1) is regulated by the carbon source used for yeast growth, responding to carbon catabolite repression. By Northern blot analyses, we determined the kinetics of glucose repression in growth-shift experiments. When added to induced cells, glucose leads to the disappearance of hybridizable aminopeptidase yscI RNA sequences within 30 min. However, the amount of inmunoreactive protein, once induced, is not affected by the addition of glucose. By deletion analysis of the fusion gene APE1-lacZ we have identified a number of strong regulatory regions in the APE1 promoter. Consensus sequences for the binding of yAP1 and the HAP2/HAP3/HAP4 complex are contained in those regions. Control of the APE1 gene expression is not mediated by the HXK2 regulatory gene, but a strain bearing a deletion in the CAT1 gene can not derepress APE1 transcription to wild-type levels.

Cis and trans-acting regulatory elements required for regulation of the CPS1 gene in Saccharomyces cerevisiae.

To clarify the transcriptional regulation by nutrient limitation of the gene encoding carboxypeptidase yscS in Saccharomyces cerevisiae (CPS1), we performed an analysis of its 5' noncoding region. In deletion experiments a sequence located between positions -644 and -591 was found to be responsible for transcriptional repression of the CPS1 gene in yeast cells grown on rich nitrogen sources. Furthermore, a 162 bp fragment spanning positions -644 to -482 of the promoter of the CPS1 gene repressed gene expression when placed 3' to the upstream activation sequence (UAS) of the heterologous gene CYC1. A fragment containing this putative upstream repression sequence (URS) was shown specifically to bind protein from a yeast extract as demonstrated by gel retardation experiments. Although a sequence mediating the control of gene expression by GCN4 was found within the URS element, the GCN4 gene product is not required for DNA-binding activity. In addition, at least three other upstream activation UASs responsible for the activation of CPS1 expression by glucose under nitrogen starvation conditions were found to be located between positions -673 and -644, -482 and -353, and -243 and -186, respectively. The putative mechanism of the nitrogen limitation-dependent regulation of CPS1 expression via these regulatory elements is discussed.

Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation.

Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium-glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide-insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent. Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine. Analysis of CPS1 expression in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat-shocked cells (38 degrees C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway.

Molecular cloning and sequencing of genomic DNA encoding yeast vacuolar carboxypeptidase yscS.

A Saccharomyces cerevisiae genomic DNA encoding vacuolar carboxypeptidase yscS was cloned from a yeast YEp13 library by complementation of the previously characterized mutation cps1-1 [(1981) J. Bacteriol. 147, 418-426], by means of staining carboxypeptidase activity in yeast colonies. The nucleotide sequence of the cloned gene was determined. The open reading frame of CPS1 consists of 576 codons and therefore encodes a protein of 64961 molecular weight. A stretch of 19 residues near the N-terminus of the deduced polypeptide sequence contains characteristics common to known hydrophobic leader sequences. CPS1 was determined by DNA blot analysis to be a single copy gene located on chromosome X. The cloned fragment was used to identify a 2.1 kb mRNA. A transcriptional activation of CPS1 occurs when cells grow on a substrate of carboxy-peptidase yscS as sole nitrogen source.

[Descriptive analysis of demand of home visits at the Pozuelo de Alarcón health center (1985-1987)]. / Análisis descriptivo de las visitas domiciliarias a demanda del Centro de Salud de Pozuelo de Alarcón (1985-1987).

The characteristics of the population cared for in home visits from 1985 to 1987 and the justification for these visits were evaluated. Elderly patients, females and consultations for respiratory diseases were more common. The patients belonging to higher socioeconomic classes were more common in the age groups below 20 years, whereas those from lower classes were more common in patients over 70 years. 70% of consultations were considered as non justified; these predominated in younger patients and in those from higher socioeconomic classes (a probably increasing group of population in Pozuelo) and from autumn to spring. Their number decreased in a near parallel fashion to that of justified consultations, probably in relation with a task of health education. We think that the population should be taught the proper use of this type of health attention, which would result in a better quality of care.