Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha.

Gietl, Christine; Faber, Klaas Nico; Klei, van der Ida; Veenhuis, Marten

doi:10.1073/pnas.91.8.3151

Cited by 141 publications

(86 citation statements)

References 25 publications

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“…In summary, we have shown that the N-terminal presequence of peroxisomal thiolase does not function for import of proteins into T brucei glycosomes, although this N-terminal peroxisomal targeting signal has been well conserved between mammalian cells, plants and fungi [17,18,24,25]. However, we cannot rule out the possibility that a related variant of this signal also functions for import of some proteins into the glycosomes of 7: brucei .…”

Section: The C-terminal Tripeptide Of Pepck Is Required Jbr Ef$cient mentioning

confidence: 93%

Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C‐terminal sequence

Sommer¹,

Nguyen²,

Wang³

1994

FEBS Letters

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Import of proteins into the glycosomes of 7Y brucei resembles the peroxisomal protein import in that C-terminal SKL-like tripeptide sequences can function as targeting signals. Many of the glycosomal proteins do not, however, possess such C-terminal tripeptide signals. Among these, phosphoenolpyruvate carboxykinase (PEPCK (ATP)) was thought to be targeted to the glycosomes by an N-terminal or an internal targeting signal. A limited similarity to the N-terminal targeting signal of rat peroxisomal thiolase exists at the N-terminus of 7: brucei PEPCK. However, we found that this peroxisomal targeting signal does not function for glycosomal protein import in Z brucei. Further studies of the PEPCK gene revealed that the C-terminus of the predicted protein does not correspond to the previously deduced protein sequence of 472 amino acids due to a -1 frame shift error in the original DNA sequence. Readjusting the reading frame of the sequence results in a predicted protein of 525 amino acids in length ending in a tripeptide serine-arginine-leucine (SRL), which is a potential targeting signal for import into the glycosomes. A fusion protein of firefly luciferase, without its own C-terminal SKL targeting signal, and 7: brucei PEPCK is efficiently imported into the glycosomes when expressed in procyclic trypanosomes. Deletion of the C-terminal SRL tripeptide or the last 29 amino acids of PEPCK reduced the import only by about 50% while a deletion of the last 47 amino acids completely abolished the import. These results suggest that 7: brucei PEPCK may contain a second, internal glycosomal targeting signal upstream of the C-terminal SRL sequence.

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Section: The C-terminal Tripeptide Of Pepck Is Required Jbr Ef$cient mentioning

confidence: 93%

Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C‐terminal sequence

Sommer¹,

Nguyen²,

Wang³

1994

FEBS Letters

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“…Methanol oxidase gene MOX promoter (MOXp) was amplified from plasmid pHIPX4 (Gietl et al 1994) using primers MOXp-L (5′-TCAAGATCTTCGACGCGGAGAACGA TCT-3′, BglII) and MOXp-R (5′-CCGAAGCTTTGTTTT TGTACTTTAGATTGATG-3′, HindIII). The BglII-EcoRI digested plasmid pGAPZα-A was ligated with the BglIIHindIII digested MOXp and HindIII-EcoRI digested α-MF.…”

Section: Construction Of Recombinant Expression Plasmidmentioning

confidence: 99%

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene

Chen

Wang

et al. 2008

Appl Microbiol Biotechnol

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Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.

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“…The mutation was introduced into the AOX gene using primers AOX G15A and AOX-downstream (Table 2). The PCR product was cloned into plasmid pHIPX4 (Gietl et al, 1994). The resulting plasmid, designated pHIPX4-AOX G15A, was linearized and integrated into the AOX locus of WT NCYC 495 as detailed above.…”

Section: Construction Of Aox Mutantsmentioning

confidence: 99%

Routing ofHansenula polymorphaAlcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

Gunkel

Dijk

Veenhuis

et al. 2004

MBoC

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Import of Hansenula polymorpha alcohol oxidase (AO) into peroxisomes is dependent on the PTS1 receptor, HpPex5p. The PTS1 of AO (-LARF) is sufficient to direct reporter proteins to peroxisomes. To study AO sorting in more detail, strains producing mutant AO proteins were constructed. AO containing a mutation in the FAD binding fold was mislocalized to the cytosol. This indicates that the PTS1 of AO is not sufficient for import of AO. AO protein in which the PTS1 was destroyed (؊LARA) was normally sorted to peroxisomes. Moreover, C-terminal deletions of up to 16 amino acids did not significantly affect AO import, indicating that the PTS1 was not necessary for targeting. Consistent with these observations we found that AO import occurred independent from the C-terminal TPR-domain of HpPex5p, known to bind PTS1 peptides. Synthesis of the N-terminal domain (amino acids 1-272) of HpPex5p in pex5 cells restored AO import, whereas other PTS1 proteins were mislocalized to the cytosol. These data indicate that AO is imported via a novel HpPex5p-dependent protein translocation pathway, which does not require the PTS1 of AO and the C-terminal TPR domains of HpPex5p, but involves FAD binding and the N-terminus of HpPex5p.

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Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha.

Cited by 141 publications

References 25 publications

Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C‐terminal sequence

Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C‐terminal sequence

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene

Routing ofHansenula polymorphaAlcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

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