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Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum.

Agaphonov MO, Romanova NV, Trushkina PM, Smirnov VN, Ter-Avanesyan MD - BMC Mol. Biol. (2002)

Bottom Line: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway.Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree.Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Str, 15A, Moscow, 121552, Russia. aga@cardio.ru

ABSTRACT

Background: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown.

Results: We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation.

Conclusions: The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

No MeSH data available.


Related in: MedlinePlus

Halo-forming ability of S. cerevisiae (Sc) and H. polymorpha (Hp) transformants expressing different uPA variants. Yeast transformants were patched on fibrin-containing plates and incubated for 20 h (H. polymorpha) or 40 h (S. cerevisiae).
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Figure 3: Halo-forming ability of S. cerevisiae (Sc) and H. polymorpha (Hp) transformants expressing different uPA variants. Yeast transformants were patched on fibrin-containing plates and incubated for 20 h (H. polymorpha) or 40 h (S. cerevisiae).

Mentions: Similar pattern of secretion of different uPA variants was revealed in the S. cerevisiae integrants carrying a single copy of the uPA expression cassette (Figure 3), indicating applicability of the obtained results to different yeast species. However, in contrast to H. polymorpha, the transformants of S. cerevisiae bearing the expression cassettes integrated in a single copy did not accumulate uPA intracellularly. On the other hand, such accumulation took place in transformants with the multicopy uPA expression vector (data not shown). Above we demonstrated that in H. polymorpha high levels of uPA expression, inhibiting secretion, are always accompanied with its intracellular accumulation (Table 1). Basing on this, we concluded that uPA expression levels in S. cerevisiae transformants with a single copy of the uPA expression cassette were lower than those, which might inhibit its secretion.


Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum.

Agaphonov MO, Romanova NV, Trushkina PM, Smirnov VN, Ter-Avanesyan MD - BMC Mol. Biol. (2002)

Halo-forming ability of S. cerevisiae (Sc) and H. polymorpha (Hp) transformants expressing different uPA variants. Yeast transformants were patched on fibrin-containing plates and incubated for 20 h (H. polymorpha) or 40 h (S. cerevisiae).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC130179&req=5

Figure 3: Halo-forming ability of S. cerevisiae (Sc) and H. polymorpha (Hp) transformants expressing different uPA variants. Yeast transformants were patched on fibrin-containing plates and incubated for 20 h (H. polymorpha) or 40 h (S. cerevisiae).
Mentions: Similar pattern of secretion of different uPA variants was revealed in the S. cerevisiae integrants carrying a single copy of the uPA expression cassette (Figure 3), indicating applicability of the obtained results to different yeast species. However, in contrast to H. polymorpha, the transformants of S. cerevisiae bearing the expression cassettes integrated in a single copy did not accumulate uPA intracellularly. On the other hand, such accumulation took place in transformants with the multicopy uPA expression vector (data not shown). Above we demonstrated that in H. polymorpha high levels of uPA expression, inhibiting secretion, are always accompanied with its intracellular accumulation (Table 1). Basing on this, we concluded that uPA expression levels in S. cerevisiae transformants with a single copy of the uPA expression cassette were lower than those, which might inhibit its secretion.

Bottom Line: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway.Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree.Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Str, 15A, Moscow, 121552, Russia. aga@cardio.ru

ABSTRACT

Background: Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown.

Results: We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation.

Conclusions: The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

No MeSH data available.


Related in: MedlinePlus