Febs Letters

Gunkel, Katja; van der Klei, Ida J.; Barth, Gerold; Veenhuis, Marten

doi: 10.1016/S0014-5793(99)00513-Xpmid: 10356972

We have shown that peroxisomes of the yeast Yarrowia lipolytica are subject to specific degradation after exposure of acetate/oleate‐grown cells to glucose excess conditions. Electron microscopic analysis has revealed that the peroxisomes were degraded by uptake in the vacuole. Our results suggest that peroxisomes are taken up by macroautophagic processes, because sequestration of individual peroxisomes, which occurs typically at the beginning of microautophagy, was never observed. The observation that a peroxisomal amine oxidase activity is specifically induced by ethylamine was used for the development of a plate assay screening procedure to isolate peroxisome degradation‐defective mutants.

Fong, Karen P.Y; Tan, Hai-Meng

doi: 10.1016/S0014-5793(99)00520-7pmid: 10356973

A second and novel cis‐benzene dihydrodiol dehydrogenase which is able to dehydrogenate a range of cis‐dihydrodiols and other vicinal alcohols has been purified from Pseudomonas putida ML2. The enzyme is a tetramer of a polypeptide of 39 kDa in molecular mass and has a pH optimum of 9.0. Despite having a primary structure that has significant similarity to glycerol dehydrogenases, the k cat/K m value of the enzyme for cis‐benzene dihydrodiol is 4300‐fold higher compared to glycerol. The apparent K m values of the enzyme for cis‐benzene dihydrodiol and glycerol are 0.01 mM and 46 mM, respectively, and 0.22 mM for NAD+.

Yue, Bai-Gong; Akusjärvi, Göran

doi: 10.1016/S0014-5793(99)00521-9pmid: 10356974

Splicing enhancers have previously been shown to promote processing of introns containing weak splicing signals. Here, we extend these studies by showing that also ‘strong’ constitutively active introns are absolutely dependent on a downstream splicing enhancer for activity in vitro. SR protein binding to exonic enhancer elements or U1 snRNP binding to a downstream 5′ splice site serve redundant functions as activators of splicing. We further show that a 5′ splice site is most effective as an enhancer of splicing. Thus, a 5′ splice site is functional in S100 extracts, under conditions where a SR enhancer is non‐functional. Also, splice site pairing occurs efficiently in the absence of exonic SR enhancers, emphasizing the significance of a downstream 5′ splice site as the enhancer element in vertebrate splicing.

Tögel, Martin; Eichinger, René; Wiche, Gerhard; Propst, Friedrich

doi: 10.1016/S0014-5793(99)00523-2pmid: 10356975

The microtubule‐associated proteins 1B and 1A are synthesized as polyprotein precursors which are rapidly cleaved to give rise to heavy and light chains constituting the respective microtubule‐associated protein 1B or microtubule‐associated protein 1A complex. To identify domains necessary for precursor processing, we expressed microtubule‐associated protein 1B deletion mutants in fibroblasts and monitored proteolytic cleavage of the precursor proteins by immunoblot analysis. We found that a novel hydrophilic, proline‐rich 45 amino acid domain containing the cleavage site is necessary and sufficient for processing. This domain is conserved in microtubule‐associated protein 1A. Additional sequences in the N‐terminal half of the heavy chain contribute to the efficiency of cleavage.

Larsen, Jakob E.; Avvakumov, George V.; Hammond, Geoffrey L.; Vogel, Lotte K.

doi: 10.1016/S0014-5793(99)00526-8pmid: 10356976

It has been suggested that N‐glycans act as a general sorting signal for secretory proteins in MDCK cells [Scheiffele et al. (1995) Nature 378, 96–98]. Human corticosteroid binding globulin contains six consensus sites for N‐glycosylation and is known to be secreted to the apical side of MDCK cells. Our results show that wild‐type corticosteroid binding globulin is N‐glycosylated when it is recombinantly expressed in MDCK cells. Six mutants, each lacking one of the N‐glycosylation sites, and a mutant lacking all six N‐glycosylation sites were also secreted to the apical side of MDCK cells in a polarized manner. Thus, the N‐glycans on corticosteroid binding globulin do not act as an apical sorting signal in MDCK cells.

Yuan, Zheng

doi: 10.1016/S0014-5793(99)00506-2pmid: 10356977

A novel method was introduced to predict protein subcellular locations from sequences. Using sequence data, this method achieved a prediction accuracy higher than previous methods based on the amino acid composition. For three subcellular locations in a prokaryotic organism, the overall prediction accuracy reached 89.1%. For eukaryotic proteins, prediction accuracies of 73.0% and 78.7% were attained within four and three location categories, respectively. These results demonstrate the applicability of this relative simple method and possible improvement of prediction for the protein subcellular location.

Rockel, Beate; Walz, Jochen; Hegerl, Reiner; Peters, Jürgen; Typke, Dieter; Baumeister, Wolfgang

doi: 10.1016/S0014-5793(99)00431-7pmid: 10356978

Valosine‐containing protein‐like ATPase from Thermoplasma acidophilum is a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N‐ethylmaleimide‐sensitive fusion protein. We have used electron tomography to obtain low‐resolution (2–2.5 nm) three‐dimensional maps of both the whole 500 kDa complex and the N‐terminally truncated valosine‐containing protein‐like ATPase from T. acidophilum complex lacking the putative substrate binding domain.

Yan, Sen Rong; Novak, M.John

doi: 10.1016/S0014-5793(99)00539-6pmid: 10356979

Tumor necrosis factor α and fMLP can activate a broad range of cellular functions in neutrophils adherent to biological surfaces. These functions are mediated by integrins and involve the activation of tyrosine kinases. Here, we report that Pyk2, a member of the focal adhesion kinase family, was present in human neutrophils and was rapidly phosphorylated and activated following tumor necrosis factor α and fMLP stimulation in an adhesion‐dependent manner. Tyrosine phosphorylation of Pyk2 was attenuated by β2 integrin blocking with specific antibodies. The tyrosine phosphorylation of Pyk2 was downstream of protein kinases Lyn, Syk and protein kinase C and cytoskeletal organization. The activation of Pyk2 may play a role in adhesion/cytoskeleton‐associated neutrophils function.

Kampers, T.; Pangalos, M.; Geerts, H.; Wiech, H.; Mandelkow, E.

doi: 10.1016/S0014-5793(99)00522-0pmid: 10356980

In Alzheimer's disease and related dementias, human tau protein aggregates into paired helical filaments and neurofibrillary tangles. However, such tau aggregates have not yet been demonstrated in transgenic mouse models of the disease. One of the possible explanations would be that mouse tau has different properties which prevents it from aggregating. We have cloned several murine tau isoforms, containing three or four repeats and different combinations of inserts, expressed them in Escherichia coli and show here that they can all be assembled into paired helical filaments similar to those in Alzheimer's disease, using the same protocols as with human tau. Therefore, the absence of pathologically aggregated tau in transgenic mice cannot be explained by intrinsic differences in mouse tau protein and instead must be explained by other as yet unknown factors.

Mikami, Yoji; Horiike, Goh; Kuroyanagi, Masanori; Noguchi, Hiroshi; Shimizu, Masanori; Niwa, Yasuo; Kobayashi, Hirokazu

doi: 10.1016/S0014-5793(99)00489-5pmid: 10356981

Analysis of genes preferentially expressed in hairy roots caused by infection with Agrobacterium rhizogenes has provided insights into the regulation of lateral root formation. A hairy root preferential cDNA, HR7, has been cloned from hairy roots of Hyoscyamus niger. HR7 encodes a novel protein partially homologous to a metallocarboxypeptidase inhibitor and is expressed exclusively in the primordium and base of lateral roots in hairy roots. Overexpression of HR7 in transgenic roots of H. niger dramatically enhances the frequency of lateral root formation. The results of this study indicate that expression of HR7 plays a critical role in initiating lateral root formation.