Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S](2+) cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S](2+) and [2Fe-2S](2+) clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions.
- Klíčová slova
- Methylobacterium extorquens AM1, PQQ, PqqE, radical SAM enzyme,
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- endopeptidasy chemie metabolismus MeSH
- kofaktor PQQ biosyntéza MeSH
- kyslík chemie MeSH
- Methylobacterium extorquens enzymologie MeSH
- molekulární sekvence - údaje MeSH
- S-adenosylmethionin chemie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- spektroskopie Mossbauerova MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- endopeptidasy MeSH
- kofaktor PQQ MeSH
- kyslík MeSH
- PqqE protein, Methylobacterium extorquens MeSH Prohlížeč
- S-adenosylmethionin MeSH
Some G protein-coupled receptors might be spacially targetted to discrete domains within the plasma membrane. Here we assessed the localization in membrane domains of the epitope-tagged, fluorescent version of thyrotropin-releasing hormone receptor (VSV-TRH-R-GFP) expressed in HEK293 cells. Our comparison of three different methods of cell fractionation (detergent extraction, alkaline treatment/sonication and mechanical homogenization) indicated that the dominant portion of plasma membrane pool of the receptor was totally solubilized by Triton X-100 and its distribution was similar to that of transmembrane plasma membrane proteins (glycosylated and non-glycosylated forms of CD147, MHCI, CD29, CD44, transmembrane form of CD58, Tapa1 and Na,K-ATPase). As expected, caveolin and GPI-bound proteins CD55, CD59 and GPI-bound form of CD58 were preferentially localized in detergent-resistant membrane domains (DRMs). Trimeric G proteins G(q)alpha/G(11)alpha, G(i)alpha1/G(i)alpha2, G(s)alphaL/G(s)alphaS and Gbeta were distributed almost equally between detergent-resistant and detergent-solubilized pools. In contrast, VSV-TRH-R-GFP, Galpha, Gbeta and caveolin were localized massively only in low-density membrane fragments of plasma membranes, which were generated by alkaline treatment/sonication or by mechanical homogenization of cells. These data indicate that VSV-TRH-R-GFP as well as other transmembrane markers of plasma membranes are excluded from TX-100-resistant, caveolin-enriched membrane domains. Trimeric G protein G(q)alpha/G(11)alpha occurs in both DRMs and in the bulk of plasma membranes, which is totally solubilized by TX-100.
- MeSH
- buněčná membrána chemie MeSH
- buněčné kultury MeSH
- centrifugace - gradient hustoty MeSH
- detergenty MeSH
- fluorescenční spektrometrie MeSH
- imunoblotting MeSH
- kaveolin 1 MeSH
- kaveoliny chemie MeSH
- lidé MeSH
- membránové mikrodomény chemie MeSH
- oktoxynol MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 analýza chemie MeSH
- radioligandová zkouška MeSH
- receptory thyroliberinu analýza chemie MeSH
- rozpustnost MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CAV1 protein, human MeSH Prohlížeč
- detergenty MeSH
- kaveolin 1 MeSH
- kaveoliny MeSH
- oktoxynol MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
- receptory thyroliberinu MeSH
The essential gene product Prp45 (379 aa) of Saccharomyces cerevisiae is a highly conserved, but N-terminally abridged, ortholog of the human transcriptional coactivator SKIP, which is involved in TGFbeta, Notch, and steroid hormone signaling. We used a diploid strain harboring PRP45 deletion, which is inviable in the haploid, to test for complementation with the truncated versions of Prp45. The N-terminal half of the protein (aa 1 to 190), denoted as the SNW domain, was found sufficient to support the essential function. Interestingly, substituting the SNW motif itself with AAA was compatible with viability. GFP-tagged Prp45 was localized in nuclear "speckles" over a diffuse nuclear background. We further found that Prp45 activated the transcription of a reporter gene in S. cerevisiae when targeted to DNA. The observed effect relied in part upon the presence of conserved helical repeats and upon the highly charged C-terminal domain (pI = 11.3). Prp45, which lacks most of the binding motifs of the human ortholog, and whose N-terminal half is sufficient for supporting the growth of prp45 cells, might be helpful in elucidating the essential function of SNW/SKIP proteins.
- MeSH
- aktivace transkripce MeSH
- fluorescenční mikroskopie MeSH
- jaderné proteiny genetika metabolismus MeSH
- konzervovaná sekvence MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- peptidové mapování MeSH
- plazmidy MeSH
- protoonkogenní proteiny genetika metabolismus MeSH
- regulace genové exprese u hub MeSH
- reportérové geny MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- terciární struktura proteinů MeSH
- testy genetické komplementace MeSH
- transfekce metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- jaderné proteiny MeSH
- protoonkogenní proteiny MeSH
- Saccharomyces cerevisiae - proteiny MeSH
