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Jolanta Wiejak

Publications and source records attributed to Jolanta Wiejak.

9 recordsLinked to original sources
PubMed2005

Cloning of two genes encoding Rab7 in Paramecium.

Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.

Amino Acid Sequence↗
PubMed2004

Dynamin- and clathrin-dependent endocytic pathway in unicellular eukaryote Paramecium.

The first evidence of dynamin presence and its colocalization with clathrin in the compartment involved in Paramecium receptor-mediated endocytosis is presented. We identified dynamin by cloning, Western blotting, and immunodetection in confocal and electron microscopy. The partial genes, which we have designated ParDyn1 and ParDyn2, are 1091 bp long, 90% identical to one another and encode the N-terminal and middle domains of Paramecium dynamin isoform 1 and isoform 2. The deduced amino acid sequences contain all three guanosine 5'-triphosphate (GTP)-binding motifs and show 67% homology to mammalian dynamins. Antibodies generated against the cloned GTPase domain revealed dynamin association with endosomes containing transferrin, the marker of receptor-mediated endocytosis. In Western blotting a strong immunoreactive polypeptide of approximately 116 kDa, which seems to be phosphorylated, was accompanied by a faint one of approximately 90 kDa in cytosolic fraction (S2). Dynamin level was correlated with internalization of transferrin and it was significantly decreased upon inhibition of this process. Immunogold labeling in electron microscopy revealed colocalization of dynamin and clathrin in coated pits and endocytic vesicles. Moreover, the polypeptide cross-reaction with 2 different antibodies against mammalian clathrin was identified by immunoblotting. These results indicate that dynamin- and clathrin-dependent pathway exists in this evolutionary ancient cell.

Amino Acid Sequence↗
PubMed2004

Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium.

Evidence that dynamin is associated with the sequestration of the Paramecium beta(2)-adrenergic receptor (betaAR) immunoanalogue is presented. We previously reported a dramatic change in the distribution of betaAR analogue in the subcellular fractions upon isoproterenol treatment: it is redistributed from the membraneous to the cytosolic fraction, as revealed by quantitative image analysis of western blots. Here we confirm and extend this observation by laser scanning confocal and immunogold electron microscopy. In the presence of isoproterenol (10 micro mol l(-1)) betaAR translocated from the cell surface into dynamin-positive vesicles in the cytoplasmic compartment, as observed by dual fluorochrome immunolabeling in a series of the confocal optical sections. Colocalization of betaAR and dynamin in the tiny endocytic vesicles was detected by further electron microscopic studies. Generally receptor sequestration follows its desensitization, which is initiated by receptor phosphorylation by G-protein-coupled receptor kinase. We cloned and sequenced the gene fragment of 407 nucleotides homologous to the beta-adrenergic receptor kinase (betaARK): its deduced amino acid sequence shows 51.6% homology in 126 amino acids that overlap with the human betaARK2 (GRK3), and may participate in Paramecium betaAR desensitization. These results suggest that the molecular machinery for the desensitization/sequestration of the receptor immunorelated to vertebrate betaAR exists in unicellular PARAMECIUM:

Adrenergic beta-Agonists↗
PubMed2003

[Role of the adaptins, dynamin like GTP-ases and Rab proteins in metabolic disorders and various infections].

Numerous metabolic and genetic diseases are due to mutations in adaptins, dynamin-like GTP-ases or disorders in trafficking machinery mediated by Rab proteins. A great number of pathogenes including viruses (HIV, SIV), bacteria and protozoa use various elements of endocytic/trafficking machinery to get into the host cells and to make their infection successful. Their different strategies are discussed.

Animals↗
PubMed2002

Genomic structure of two ras family genes in the slime mold Physarum polycephalum.

Genomic structure of two Physarum polycephalum ras family genes, Ppras2 and Pprap1, has been determined, including the upstream region of the latter. The genes are interrupted by three and four introns, respectively. The first intron of Ppras2 has the same location within the coding sequence as the first intron in another ras homolog from this organism, Ppras1 [Trzcińska-Danielewicz, J., Kozlowski, P., and Toczko, K. (1996). "Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene, a homologue of the ras protooncogene", Gene 169, pp. 143-144]. All introns, ranging from 53 to ca. 460 base pairs, have the canonical 5' and 3' ends, are greatly enriched in pyrimidines in the coding strand and have frequent pyrimidines-only tracts. These latter features seem to be responsible for the difficulties in cloning and sequencing of parts of these genes. Short sequences shared with P. polycephalum transposon-like repeats are common in the introns, indicating a possible role of transposition in intron evolution. In all three ras family genes phase zero introns are located mostly between sequences coding for regular protein secondary structure elements.

Amino Acid Sequence↗
PubMed2002

Dynamin: characteristics, mechanism of action and function.

Dynamin - a member of the GTP-ase protein family - is essential for many intracellular membrane trafficking events in multiple endocytic processes. The unique biochemical features of dynamin - especially its propensity to assemble - enable severing the nascent vesicles from the membrane. The mechanism of dynamin's action is still a subject of debate - whether it functions as a mechanochemical enzyme or a regulatory GTPase. The GTPase domain of dynamin contains three GTP-binding motifs. This domain is very conservative across the species, including that recently cloned by us in the unicellular eukaryote Paramecium. Dynamin interacts with a number of partners such as endophilin and proteins involved in coordination of endocytosis with motor molecules. A growing body of evidence indicates that dynamin and dynamin-related proteins are involved both in pathology and protection against human diseases. The most interesting are dynamin-like Mx proteins exhibiting antiviral activity.

Actins↗
PubMedDate not supplied

Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium.

Molecular search for the homologues of the mammalian proteins in the unicellular eukaryote Paramecium involved in endocytosis and membrane trafficking is discussed. We cloned and sequenced the gene fragments encoding the following components participating in endosome formation, sorting and maturation of the proprotein precursors, respectively, dynamin 2, Rab7 and furin. There is a proof that all these genes are expressed in this unicellular organism. The function of the identified immunoanalogues of the above described components of Paramecium endocytic machinery as well as a high degree of sequence homology to the respective human counterparts points to the evolutionary conservancy of these pathways.

Animals↗
PubMedDate not supplied

Immunoanalogue of vertebrate beta-adrenergic receptor in the unicellular eukaryote Paramecium.

Cell fractionation, SDS-PAGE, quantitative Western blot, confocal immunolocalization and immunogold labelling were performed to find an interpretation of the physiological response of the unicellular eukaryote Paramecium to beta-adrenergic ligands. The 69 kDa polypeptide separated by SDS-PAGE in S2 and P2 Paramecium subcellular fractions cross-reacted with antibody directed against human beta2-adrenergic receptor. This was detected by Western blotting followed by chemiluminescent detection. Quantitative image analysis showed that beta-selective adrenergic agonist (-)-isoproterenol--previously shown to enhance phagocytic activity--evoked redistribution of the adrenergic receptor analogue from membraneous (P2) to cytosolic (S2) fraction. The relative increase in immunoreactive band intensity in S2 reached 80% and was paralleled by a 59% decrease in P2 fraction. Confocal immunofluorescence revealed beta2-adrenergic receptor sites on the cell surface and at the ridge of the cytopharynx--where nascent phagosomes are formed. This localization was confirmed by immunoelectron microscopy. These results indicate that the 69 kDa Paramecium polypeptide immunorelated to vertebrate beta2-adrenergic receptor appeared in this evolutionary ancient cell as a nutrient receptor.

Animals↗