The cytosolic glycoprotein FP21 of Dictyostelium discoideum is encoded by two genes resulting in a polymorphism at a single amino acid position

West, Christopher M.; Kozarov, Emil; Teng-umnuay, Patana

doi:10.1016/s0378-1119(97)00194-7

Cited by 20 publications

(29 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells were filterlysed, and an S100 fraction was prepared by ultracentrifugation (17). SKP1 from Ax3 and HL250 were fractionated into pools-I and -II by DEAE-anion exchange chromatography (14); SKP1-c-myc from strain HW120 eluted after pool-II. Each pool was subsequently purified by phenyl-Sepharose and monoclonal antibody 3F9 affinity chromatographies (14).…”

Section: Methodsmentioning

confidence: 99%

“…SKP1 from Ax3 and HL250 were fractionated into pools-I and -II by DEAE-anion exchange chromatography (14); SKP1-c-myc from strain HW120 eluted after pool-II. Each pool was subsequently purified by phenyl-Sepharose and monoclonal antibody 3F9 affinity chromatographies (14). For metabolic labeling, a 200-ml culture of Ax3 cells was grown for three generations in 0.05 mCi/ml [ 3 H]Fuc in HL-5.…”

Section: Methodsmentioning

confidence: 99%

“…SKP1 was isolated in the same manner except the phenyl-Sepharose step was omitted, and purified radiolabeled SKP1 was pooled with unlabeled SKP1. In some cases, SKP1 was reduced and carboxamidomethylated (14), and further purified on a C 2 /C 18 (PC 3.2/3) RP-HPLC column on an Amersham Pharmacia Biotech SmartSystem HPLC (14). The peak that contained Ͼ80% of total SKP1 was examined further.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The Cytoplasmic F-box Binding Protein SKP1 Contains a Novel Pentasaccharide Linked to Hydroxyproline inDictyostelium

Teng-umnuay

Morris²,

Dell³

et al. 1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

SKP1 is involved in the ubiquitination of certain cell cycle and nutritional regulatory proteins for rapid turnover. SKP1 from Dictyostelium has been known to be modified by an oligosaccharide containing Fuc and Gal, which is unusual for a cytoplasmic or nuclear protein.To establish how it is glycosylated, SKP1 labeled with [ 3 H]Fuc was purified to homogeneity and digested with endo-Lys-C. A single radioactive peptide was found after two-dimensional high performance liquid chromatography. Analysis in a quadrupole time-of-flight mass spectrometer revealed a predominant ion with a novel mass. Tandem mass spectrometry analysis yielded a set of daughter ions which identified the peptide and showed that it was modified at Pro-143. A second series of daughter ions showed that Pro-143 was hydroxylated and derivatized with a potentially linear pentasaccharide, Hex3 Hex3 Fuc3 Hex3 HexNAc3(HyPro). The attachment site was confirmed by Edman degradation. Gas chromatography-mass spectrometry analysis of trimethylsilyl-derivatives of overexpressed SKP1 after methanolysis showed the HexNAc to be GlcNAc. Exoglycosidase digestions of the glycopeptide from normal SKP1 and from a fucosylation mutant, followed by matrix-assisted laser desorption time-of-flight mass spectrometry analysis, showed that the sugar chain consisted of D-Galp␣136-D-Galp␣13L-Fucp␣132-DGalp␤133GlcNAc. Matrix-assisted laser-desorption timeof-flight mass spectrometry analysis of all SKP1 peptides resolved by reversed phase-high performance liquid chromatography showed that SKP1 was only partially hydroxylated at Pro-143 and that all hydroxylated SKP1 was completely glycosylated. Thus SKP1 is variably modified by an unusual linear pentasaccharide, suggesting the localization of a novel glycosylation pathway in the cytoplasm.SKP1 is found in a multiprotein complex with cullin (a cdc53 homologue) and an F-box-containing protein to form the SCF complex, named as an acronym of the participating proteins. When this complex contains an E2 enzyme, it is responsible for ubiquitinating various target proteins, depending on the identity of the F-box protein. Targets for subsequent degradation identified in Saccharomyces cerevisiae include cell cycle proteins such as the S-phase kinase inhibitor SIC1 and G 1 cyclins, and proteins specific to the nutrition of the cell (1-4). The SCF complex has also been implicated in phosphorylation of kinetochore proteins (5), and another distantly related complex affects mRNA metabolism (6). An SCF complex with Cyclin A and Cdk2 has been detected in mammalian cells, and its abundance appears increased in transformed cells (7,8). SKP1 itself is abundantly and dynamically expressed in the mouse embryo (9) and central nervous system including postmitotic neurons (10) and at very high concentrations in the inner ear organ of Corti (11,12), where it comprises up to 5% of total protein in the cytoplasm. The expression of several SKP1 genes in plants appears to be governed by morphogenetic boundaries (13). Thus SKP1 is expressed ubiquitously ...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Cytoplasmic F-box Binding Protein SKP1 Contains a Novel Pentasaccharide Linked to Hydroxyproline inDictyostelium

Teng-umnuay

Morris²,

Dell³

et al. 1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…These intermediate glycoforms of Skp1A1(HW120)-myc are potentially useful acceptor substrates for assaying the activity of the GalTs whose substrates are accumulated, as found for the Skp1 GlcNAcT activity (11). Although the function of the pe-ripheral ␣-Gals is not known, they appear to be heterogeneous between two normal cellular pools of Skp1 (17).…”

mentioning

confidence: 99%

Specificity of a Soluble UDP-Galactose:Fucoside α1,3-Galactosyltransferase That Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium

Ketcham

Wang

Fisher

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Skp1 is an adaptor-like protein in E3SCF -ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Gal␣1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn 2؉ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Gal␣1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to ␣1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K m and 35-fold higher V max compared with a simple ␣-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V max advantage, but still much less active than Skp1 itself because of a K m difference. These findings indicate that ␣-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Gal␣1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

show abstract

“…Each of the major components of the yeast and mammalian SCF1 ubiquitin ligase complex has been identified in D. discoideum, appears to function in a manner identical to those in yeast and mammalian cells, and represents a key pathway for posttranslational modification of proteins (7,12,25,26,38,44,47). Protein ubiquitination has been also characterized in the ciliate Tetrahymena and Acanthamoeba (19,32), and both are permissive protozoan hosts for L. pneumophila (14).…”

mentioning

confidence: 99%

Indispensable Role for the Eukaryotic-Like Ankyrin Domains of the Ankyrin B Effector ofLegionella pneumophilawithin Macrophages and Amoebae

Price¹,

Al‐Khodor²,

Al-Quadan³

et al. 2010

Infect Immun

View full text Add to dashboard Cite

The Dot/Icm-translocated ankyrin B (AnkB) effector of Legionella pneumophila exhibits molecular mimicry of eukaryotic F-box proteins and is essential for intracellular replication in macrophages and protozoa. In addition to two eukaryotic-like ankyrin (ANK) domains, AnkB harbors a conserved eukaryotic F-box domain, which is involved in polyubiquitination of proteins throughout the eukaryotic kingdom. We have recently shown that the F-box domain of the AnkB effector is essential for decoration of the Legionella-containing vacuole (LCV) with polyubiquitinated proteins within macrophages and protozoan hosts. To decipher the role of the two ANK domains in the function of AnkB, we have constructed in-frame deletion of either or both of the ANK domain-encoding regions (ankB⌬A1, ankB⌬A2, and ankB⌬A1A2) to trans-complement the ankB null mutant. Deletion of the ANK domains results in defects in intracellular proliferation and decoration of the LCV with polyubiquitinated proteins. Export of the truncated variants of AnkB was reduced, and this may account for the observed defects. However, while full-length AnkB ectopically expressed in mammalian cells trans-rescues the ankB null mutant for intracellular proliferation, ectopic expression of AnkB⌬A1, AnkB⌬A2, and AnkB⌬A1A2 fails to trans-rescue the ankB null mutant. Importantly, ectopically expressed full-length AnkB is targeted to the host cell plasma membrane, where it recruits polyubiquitinated proteins. In contrast, AnkB⌬A1, AnkB⌬A2, and AnkB⌬A1A2 are diffusely distributed throughout the cytosol and fail to recruit polyubiquitinated proteins. We conclude that the two eukaryotic-like ANK domains of AnkB are essential for intracellular proliferation, for targeting AnkB to the host membranes, and for decoration of the LCV with polyubiquitinated proteins.

show abstract

The cytosolic glycoprotein FP21 of Dictyostelium discoideum is encoded by two genes resulting in a polymorphism at a single amino acid position

Cited by 20 publications

References 23 publications

The Cytoplasmic F-box Binding Protein SKP1 Contains a Novel Pentasaccharide Linked to Hydroxyproline inDictyostelium

The Cytoplasmic F-box Binding Protein SKP1 Contains a Novel Pentasaccharide Linked to Hydroxyproline inDictyostelium

Specificity of a Soluble UDP-Galactose:Fucoside α1,3-Galactosyltransferase That Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium

Indispensable Role for the Eukaryotic-Like Ankyrin Domains of the Ankyrin B Effector ofLegionella pneumophilawithin Macrophages and Amoebae

Contact Info

Product

Resources

About