The leucine-based sorting motifs in the cytoplasmic domain of the invariant chain are recognized by the clathrin adaptors AP1 and AP2 and their medium chains

Recognition of sorting signals within the cytoplasmic tail of membrane proteins by adaptor protein complexes is a crucial step in membrane protein sorting. The three known adaptor complexes, AP1, AP2, and AP3, have all been shown to recognize tyrosine- and leucine-based sorting signals, which are the most common sorting signals within membrane protein cytoplasmic tails. Although tyrosine-based signals are recognized by the micro-chains of adaptor complexes, the subunit recognizing leucine-based sorting signals is less clear. In this report we show by surface plasmon resonance that the two leucine-based sorting signals within the cytoplasmic tail of the invariant chain bind independently from each other to AP1 and AP2 but not to AP3. We also show that both motifs can be recognized by the micro-chains of AP1 and AP2. Moreover, by using monomeric as well as trimeric invariant chain constructs, we show that adaptor binding does not require trimerization of the invariant chain.     

Structural requirements for interactions between leucine-sorting signals and clathrin-associated adaptor protein complex AP3

Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals, but the invariant chain interacts only with AP1 and AP2, whereas LIMPII interacts strongly with AP3. In a series of in vitro experiments, we investigated the effect of residues upstream of the leucine pairs and demonstrated that these residues determine adapter binding, and certain residues favor interactions with AP3. Furthermore, constructs that interacted stronger with AP3 interacted weakly with AP1 and vice versa. Exchanging residues upstream of the leucine-based signal in LIMPII with those of the invariant chain reduced LIMPII binding to AP3 in vitro, and in vivo the corresponding LIMPII mutant was rerouted via the plasma membrane like the invariant chain. These preferential interactions of different leucine signals with different AP complexes may thus be the determining step sorting proteins from the trans-Golgi network to their final destinations. Proteins that interact with AP3 are sorted directly to endosomes/lysosomes, whereas proteins that interact with AP1 are sorted via a different route. At the same time, constructs that exhibited specificity for either AP1 or AP3 might still interact with AP2, suggesting that AP2 may recognize a wider variety of leucine signals. This is consistent with the suggested role of AP2 in internalization of proteins containing general leucine-based signals, including proteins that have been missorted to the plasma membrane.     

Host specificity dynamics: observations on gyrodactylid monogeneans

The directly transmitted viviparous gyrodactylids have high species richness but low morphological and biological diversity, and many species are recorded from only a single host. They therefore constitute a guild of species ideal for studies of the evolutionary significance of host specificity. The group has the widest host range of any monogenean family, being found on 19 orders of bony fish. However, individual species range from narrowly specific (71% of 402 described species recorded from a single host) to extremely catholic (Gyrodactylus alviga recorded from 16 hosts). Gyrodactylid-host interactions extend from 60 mya (G. lotae, G. lucii) down to 150 years (G. derjavini on Oncorhynchus mykiss). Co-evolution with the host is comparatively rare within the gyrodactylids, but host switching or ecological transfer is common, and has been facilitated by the mixing of fish strains that followed glaciation. In this review, we consider the factors responsible for gyrodactylid specificity patterns, using examples from our work on salmonid gyrodactylids including G. salaris, responsible for major epidemics on wild Atlantic salmon (Salmo salar) in Norway since 1975, and G. thymalli from grayling and G. derjavini from trout.G. salaris has a wide host range with highest population growth rates on Norwegian salmon strains. However, growth rates are variable on both host strains and species, because of the multitude of micro- and macro-environmental factors influencing parasite mortality and fecundity. A better predictor of performance is the proportion of fishes of a strain which are innately resistant to the parasite, a measure which is negatively correlated with the time to peak infection in a host strain. Population growth rate is also negatively correlated with age of infection; the initial rate, therefore, predicts best the suitability of a fish as host for G. salaris. The host response to gyrodactylids appears to be the same mechanism in all salmonids with innate resistance as one end of a spectrum, but influenced by stress and probably under polygenic control. Hybrid experiments show that performance of G. salaris on a host is heritable, and usually intermediate between that of the parents. This host response mechanism, coupled with the initial parasite population growth on a fish, determines the host specificity, i.e. whether the fish will be susceptible, a responder or innately resistant. The use of population growth rate parameters allows comparison of different hosts as a resource for a gyrodactylid. In the case of G. salaris, East Atlantic and Baltic strains of Atlantic salmon are core hosts, but other salmonids can physiologically sustain infections for considerable periods, and may be important in parasite dispersal and transmission. A further group of non-salmonid fishes are unable to sustain G. salaris reproduction, but can act as transport hosts.Population growth parameters are very labile to stressors and environmental factors, particularly temperature and salinity, and also other aspects of host ecology and water quality. These factors may also influence the spectrum of hosts that can be infected under particular conditions, and probably favoured ecological transfer of gyrodactylids between host species in periglacial conditions. G. salaris may still be undergoing post-glacial range expansion (aided by anthropogenic spread) as shown by the increase in the species range over the last 25 years. The origin of G. salaris, G. teuchis and G. thymalli is discussed in relation to glacial refugiums during the last ice age.     

The host specificity of Gyrodactylus salaris Malmberg (Platyhelminthes, Monogenea): susceptibility of Oncovhynchus mykiss (Walbaum) under experimental conditions

An experimental epidemiological approach was chosen to study the survival and infection dynamics of Gyrodactylus salaris on juvenile rainbow trout, Oncorhynchus mykiss , in the laboratory. A marked heterogeneity in the host stock was apparent. The rainbow trout could be divided into three groups on the basis of parasite survival and infection pattern on individually isolated fish: (1) hosts receptive to initial parasite attachment, but unreceptive to parasite establishment and reproduction; (2) hosts moderately susceptible to parasite establishment and reproduction, but which, after a period of restricted parasite population growth, responded, recovered and eliminated the parasites; and (3) hosts very susceptible to parasite infection and reproduction, but which, after a period of significant parasite population growth, responded, recovered and eliminated the parasites. These different patterns are considered to reflect genetic differences between host individuals. Parasite aggregation was also shown to be an important factor in the outcome of the host-parasite association. The parasites were finally eliminated on the individually isolated hosts, but not on hosts maintained in batches and so host population size and immigration of fresh. previously unexposed, hosts appeared to be important for growth and maintenance of the parasite population. The parasite was not found to cause host mortality. Rainbow trout was a suitable host for G. salaris , capable of transmitting the parasite to new localities as a consequence of stocking programmes or migratory behaviour.     

Nuclear and mitochondrial forms of human uracil-DNA glycosylase are encoded by the same gene.

Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800. This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting. To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration. An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.     

Binding of intrinsic factor to ileal brush border membrane in the rat

The rat ileal brush border membrane binds both free [¹²⁵I]-intrinsic factor (IF) and the IF-[⁵⁷Co]cobalamin (cbl) complex. This binding is observed with IF isolated from rat stomach, but not from IF isolated from hog, canine and human stomachs. The binding of rat-IF[⁵⁷Co]cbl can be blocked with free rat IF but not with hog IF. The IF-cbl complex binds at a higher affinity (Ka=0.15 × 10⁹ M^?1) compared to that of free IF (Ka=0.9 × 10⁹ M^?1). Rat IF-cbl also binds efficiently to human and canine ileal membranes. While antibody to the canine ileal receptor blocks the binding of rat, human or hog IF-[⁵⁷Co]cbl to human and canine ileal membranes, it does not affect the binding of rat IF-[⁵⁷Co]cbl to rat ileal membranes. These findings demostrate that the rat ileal receptor is different from canine and human ileal receptors.     

Taxonomy of Leucochloridium sp. (Digenea) infecting Succinea pfeifferi Rossm?ssler, 1835

The anatomical and morphological variability of sporocysts and metacercariae of Leucochloridium sp. recovered from natural infected Succinea pfeifferi Rossm?ssler collected in the Agdenes area. Norway (63 degrees 35'N, 9 degrees 45'E), was studied by light microscopy. The results are compared with five species from the Nearctic and Palearctic with known adult stage (L. variae McIntosh, 1932; L. fuscostriatum Robinson, 1947; L. perturbatum Pojmanska, 1969; L. subtilis Pojmanska, 1969; L. fuscum Rietschel, 1970) and nine species with unknown adult stage. In the larval stages no morphological taxonomical characters were found to separate Leucochloridium sp. as a species distinct from the five (and nine) species. The variability and validity of the characters used is discussed.     

Using genomics to guide seed‐sourcing at the right taxonomical level for ecological restoration projects: The complex case of Carex bigelowii s.lat. in Norway

There is a growing demand for ecological restoration using suitable seeds following international standards or national legal demands for local seed‐sourcing. However, before selecting the appropriate geographic origin of seeds, it is vital to explore taxonomic complexity related to the focal taxa. We used ddRAD‐seq to screen genomic diversity within Carex bigelowii s.lat. focussing on Norway. This species complex is considered a candidate for seeding, but presents considerable morphological, ecological, and genetic variation. The genetic structure of 132 individuals of C. bigelowii s.lat., including Carex nigra as an outgroup, was explored using ordinations, clustering analyses, and a genetic barrier algorithm. Two highly divergent clusters were evident, supporting the recognition of two taxonomic units “C. dacica” and C. bigelowii “subsp. bigelowii”. Previously defined seed‐sourcing regions for C. bigelowii s.lat. did not consider the known taxonomic complexity, and therefore interpreted the overall genetic structure as seed‐sourcing regions, not taxa. We estimated genetic neighborhood sizes within each taxon to be 100–150 km and 300 km, respectively, indicating species‐specific delimitations of local seed‐sourcing regions. Frequent hybrids, local genetic distinctiveness, and suggested ecotypes add complexity to the discussed seed‐sourcing regions. Our results show how genomic screening of diversity and structure in a species complex can alleviate the taxonomic impediment, inform practical questions, and legal requirements related to seed‐sourcing, and together with traditional taxonomic work provide necessary information for a sound management of biodiversity.     

The extreme Beringian/Atlantic disjunction in Saxifraga rivularis (Saxifragaceae) has formed at least twice

Aim The oceanic Saxifraga rivularis L. presents one of the most extreme disjunctions known in the arctic flora: it has a small amphi‐Beringian range and a larger amphi‐Atlantic one. It was recently suggested to have had a single allopolyploid origin in Beringia at least one glacial cycle ago, followed by gradual expansion in a more humid period and differentiation into two allopatric subspecies (the Atlantic ssp. rivularis and the Beringian ssp. arctolitoralis). Here we explore the history of its extreme disjunction. Location The amphi‐Beringian and northern amphi‐Atlantic regions. Methods We obtained amplified fragment length polymorphisms (AFLPs) and chloroplast DNA sequences from 36 populations (287 individuals) and 13 populations (15 individuals), respectively. The data were analysed using principal coordinates analyses, Bayesian clustering methods, and analyses of molecular variance. Results Two distinctly divergent AFLP groups were observed, corresponding to the two described subspecies, but, surprisingly, four of the West Atlantic populations belonged to the supposedly Beringian endemic ssp. arctolitoralis. This was confirmed by re‐examination of their morphological characteristics. The overall AFLP diversity in the species was low (26.4% polymorphic markers), and there was no variation in the five investigated chloroplast DNA (cpDNA) regions. There was little geographic structuring of the AFLP diversity within each subspecies, even across the extreme disjunction in ssp. arctolitoralis, across the Bering Sea, and across the Atlantic Ocean, except that most plants from the arctic Svalbard archipelago formed a separate genetic group with relatively high diversity. Main conclusions The extreme disjunction in S. rivularis has evidently formed at least twice. The first expansion from Beringia was followed by allopatric differentiation into one Beringian and one Atlantic subspecies, which are distinctly divergent at AFLP loci but still harbour identical cpDNA haplotypes, suggesting that the expansion was quite recent but before the last glaciation. The next expansion from Beringia probably occurred by means of several long‐distance dispersals in the current interglacial, resulting in the colonization of the western Atlantic region by ssp. arctolitoralis. The poor geographic structuring within each subspecies suggests frequent long‐distance dispersals from two main Weichselian refugia, one Beringian and one western‐central European, but it is possible that the genetic group in Svalbard originates from an additional refugium.