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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

The aggregation pattern of uPA-KDEL in the opu24 mutant. Western blot analysis of uPA. S and P, the supernatant and pellet fractions of the cell lysates of 24–8 V/pNR4 (opu24) and 8 V/pNR4 (OPU24) strains. Pellets were dissolved in a 100-fold volume of starting lysate.
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Figure 5: The aggregation pattern of uPA-KDEL in the opu24 mutant. Western blot analysis of uPA. S and P, the supernatant and pellet fractions of the cell lysates of 24–8 V/pNR4 (opu24) and 8 V/pNR4 (OPU24) strains. Pellets were dissolved in a 100-fold volume of starting lysate.

Mentions: Above it was shown that accumulation of uPA within the ER causes its aggregation. This means that defect of degradation of uPA in this compartment should increase its amount and, therefore, the aggregation degree. This suggestion was supported by the results of analysis of the aggregation pattern of uPA-KDEL expressed in the H. polymorpha opu24 mutant. The uPA supersecretion opu24 mutation has been previously shown to cause phenotypes characteristic of the ERAD lesion [10]. Impairment of the ERAD should save misfolded uPA from degradation, thus increasing its amount within the ER. The amount of aggregated but not soluble form of uPA-KDEL appeared to be much higher in the mutant than in the wild type cells confirming that accumulation of uPA in the ER is accompanied by its aggregation (Figure 5). Interestingly, this mutation did not noticeably influence the aggregation pattern of the full-length uPA lacking the ER retention signal (data not shown). This indicates that accumulation of wild type uPA within the ER of the mutant cells was not accelerated though degradation of misfolded protein was impaired. Thus, it is possible to suggest that this mutation facilitates exit of uPA from the ER probably due to the activation of mechanisms improving efficiency of its folding.


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)

The aggregation pattern of uPA-KDEL in the opu24 mutant. Western blot analysis of uPA. S and P, the supernatant and pellet fractions of the cell lysates of 24–8 V/pNR4 (opu24) and 8 V/pNR4 (OPU24) strains. Pellets were dissolved in a 100-fold volume of starting lysate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: The aggregation pattern of uPA-KDEL in the opu24 mutant. Western blot analysis of uPA. S and P, the supernatant and pellet fractions of the cell lysates of 24–8 V/pNR4 (opu24) and 8 V/pNR4 (OPU24) strains. Pellets were dissolved in a 100-fold volume of starting lysate.
Mentions: Above it was shown that accumulation of uPA within the ER causes its aggregation. This means that defect of degradation of uPA in this compartment should increase its amount and, therefore, the aggregation degree. This suggestion was supported by the results of analysis of the aggregation pattern of uPA-KDEL expressed in the H. polymorpha opu24 mutant. The uPA supersecretion opu24 mutation has been previously shown to cause phenotypes characteristic of the ERAD lesion [10]. Impairment of the ERAD should save misfolded uPA from degradation, thus increasing its amount within the ER. The amount of aggregated but not soluble form of uPA-KDEL appeared to be much higher in the mutant than in the wild type cells confirming that accumulation of uPA in the ER is accompanied by its aggregation (Figure 5). Interestingly, this mutation did not noticeably influence the aggregation pattern of the full-length uPA lacking the ER retention signal (data not shown). This indicates that accumulation of wild type uPA within the ER of the mutant cells was not accelerated though degradation of misfolded protein was impaired. Thus, it is possible to suggest that this mutation facilitates exit of uPA from the ER probably due to the activation of mechanisms improving efficiency of its folding.

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