Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum
© Agaphonov et al; licensee BioMed Central Ltd. 2002
Received: 12 March 2002
Accepted: 7 October 2002
Published: 7 October 2002
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.
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.
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.
Though the secretory pathway is organized similarly in yeast and in other eukaryotic organisms, not all proteins of higher eukaryotes can be efficiently secreted from yeast cells. Secretion of some recombinant or mutant proteins is affected by their improper folding in the yeast endoplasmic reticulum (ER) [1, 2] and rapid degradation of misfolded molecules by cytosolic proteasome complex after retrograde translocation from the lumenal to cytoplasmic face of the ER (ER associated protein degradation, ERAD) [3, 4]. Alternatively, misfolded proteins can be sorted from the late Golgi to vacuole, yeast homologue of lysosome, and degraded by the vacuolar protease complex [5, 6]. The recognition and retention of misfolded proteins in the ER is carried out by the "ER quality control" (ERQC) machinery. Saccharomyces cerevisiae differs by the organization of ERQC not only from higher eukaryotes, but also from Schizosaccharomyces pombe. However, in spite of these species-specific differences, the ERQC involves processing of the N-linked oligosaccharides in all eukaryotes reflecting importance of N-glycosylation for protein folding in vivo [for review, see ].
Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast. Although three mutations improving uPA secretion in yeast have been characterized [8–10], the bottleneck for uPA secretion remains uncertain. There are some data indicating that the efficiency of uPA secretion in yeast depends on the ERQC machinery. Disruption of the S. cerevisiae PMR1 gene, encoding the Golgi membrane Ca2+ ion pump, improves uPA secretion, decreases protein glycosylation  and causes a defect of the ERAD . The Hansenula polymorpha opu24 mutation with the uPA supersecretion phenotype also impairs protein glycosylation and possibly causes defect of the ERAD, since, similarly to pmr1, the opu24 mutant is also hypersensitive to dithiothreitol and tunicamycin, drugs perturbing protein folding in the ER . However, the S. cerevisiae ssu21 mutation improving uPA secretion, does not alter neither glycosylation of secretory proteins , nor sensitivity to dithiothreitol and tunicamycin (M. Agaphonov, unpublished).
In this study we demonstrate that inefficient uPA secretion in yeast is related to its retention in the ER that is conditioned by the uPA N-terminal domains. Accumulation of uPA within the ER affects its folding and leads to formation of high molecular weight aggregates.
Overexpression of the uPA impairs its secretion and causes accumulation in an aggregated form
Dependence of u-PA levels on the copy number and integration locus of the expression cassette.
Copy number in locus:
activity in culture medium
activity in cell lysate
amount in cell lysate*
In contrast to the extracellular form, intracellular uPA was core glycosylated, since it migrated on SDS gel as a distinct band and its electrophoretic mobility only slightly increased after EndoH treatment (Figure 2, lanes 1 and 2). This means that the N-linked oligosaccharide chain of intracellular uPA had not received the Golgi modifications, and, therefore, uPA aggregates were formed within the ER. This conclusion was further confirmed (see below).
Secretion of uPA depends on the presence of N-glycosylation site and N-terminal domains
Comparison of secretion efficiencies of different uPA variants by H. polymorpha
Amount in culture medium determined by:
Fibrinolytic activity assay (%)
Western blotting* (%)
Aggregation of uPA is defined by accumulation within the ER
Distribution of different uPA variants between the soluble and pellet fractions obtained by centrifugation of cell lysates (Figure 1) depended on their secretion potential. The highest proportion of soluble to aggregated form was observed for the best secreted uPA variant (uPA-C), whereas the lowest one was in the case of the worst secreted variant (uPA-Q). Since the major bands of the soluble fractions of the uPA and uPA-C variants corresponded to the core-glycosylated protein, they might represent a pool of newly translocated molecules, whose fate had not been determined yet, and properly folded molecules, which are ready to leave the ER or already entered the Golgi apparatus, but have not received modifications altering their electrophoretic mobility.
Accumulation of the wild type uPA in the H. polymorpha ER may result from its retention in this compartment due to incorrect folding in the heterologous host. Normally misfolded proteins in the ER are eliminated by the ERAD pathway. The data presented in this study suggest that uPA can partially degrade within the yeast cell. In fact, the rate of uPA synthesis in the transformant DLU/pAM226, bearing two copies of the expression cassette, must not exceed more than two-fold the rate of uPA synthesis in the DLU/L integrant with a single copy of this cassette. However, the difference in amount of intracellular uPA in these transformants appeared to be much bigger (Table 1). The discrepancy between the expected and observed difference in intracellular accumulation of uPA cannot be explained by worsening uPA secretion, since in the DLU/L strain the amount of extracellular enzyme constituted less than 10% of that of its intracellular form (0.11 μg of extracellular uPA vs. 1.6 μg of intracellular uPA per 1 mg of total cellular protein). Thus, the observed difference in amount of intracellular uPA probably was due to the degradation of significant portion of uPA in the transformants with lower expression levels and rescue of misfolded uPA by aggregation upon the higher expression levels.
The data obtained show that efficient uPA secretion demands an optimal level of its expression: both low and increased expression did not result in the maximal levels of uPA secretion. Indeed, we observed that the best uPA secreting strains were those containing the uPA expression cassette integrated in a single copy into the MOX or LEU2 genes. In contrast, integrants carrying a single copy of the uPA cassette integrated into a telomere region produced less uPA and were less efficient in uPA secretion. On the other hand, strains with multiple copies of the integrated cassette also secreted less uPA and accumulated it in an inactive form intracellularly. Comparison of strains with uPA expression exceeding an optimal level showed an inverted correlation between the efficiency of its expression and secretion (Table 1). This can explain why no mutants with increased uPA expression levels were obtained so far among those producing more of its extracellular form.
We also found that upon overexpression uPA is accumulated in a form of high molecular weight aggregates. The absence of the Golgi modifications in accumulated uPA suggested that its aggregation occurred within the ER. Deletion analysis has shown that the N-terminal domains of uPA were responsible for the elevated levels of its aggregation. Alteration of the N-glycosylation site of uPA also resulted in its intracellular accumulation and aggregation. Thus, it is possible to suggest that both the presence of N-terminal domains and the absence of N-glycosylation caused intracellular accumulation of uPA, which interfered with its proper folding and resulted in subsequent aggregation in the ER. In agreement with this, the presence of the ER retention signal significantly increased aggregation degree of the best secreted uPA variant lacking the N-terminal domains.
Some of the ERAD substrates, e. g. mutant carboxy peptidase Y, can escape degradation and accumulate within the ER if they lack the N-linked oligosaccharide chains . Thus, it is possible that the misfolded uPA variants lacking the N-glycosylation site also escape degradation and accumulate in the ER, interfering with the folding of newly synthesised molecules due to the "crowding" effect. This may explain why these uPA variants demonstrated worse secretion and higher propensity to aggregate.
The aggregation of uPA on its own is unlikely to be the cause of its poor secretion by yeast cells, since transformants bearing a single copy of the uPA expression cassette integrated into the MOX or LEU2 loci showed both higher secretion rate of uPA and higher degree of its aggregation, than transformant with a single copy of this cassette integrated into a telomere region. Moreover, it is possible that to some extent, the aggregation of uPA even improves its secretion: accumulation in aggregates may decrease uPA crowding in the ER, thus facilitating its proper folding and subsequent secretion.
The increased aggregation of uPA-KDEL in the H. polymorpha opu24 mutant provides an additional evidence for the interference of uPA accumulation within the ER with its folding. We suggest that the degradation rate of uPA-KDEL in this mutant was reduced due to the ERAD lesion. In contrast, the opu24 mutation did not stimulate aggregation of uPA lacking the ER retention signal. This indicates that a significant portion of uPA escaped from the ER, probably due to improved folding, which may be the reason of the "supersecretion" phenotype of this mutant.
In this work we demonstrate that in H. polymorpha overexpression poisons uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that the aggregation occurs within the ER. Deletion analysis has shown that the N-terminal domains of uPA were responsible for its poor 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 ER by means of the KDEL signal also led to its accumulation in an aggregated, but not in soluble form, and the amount of uPA-KDEL aggregates increased in the opu24 mutant with defects in the ER-associated degradation of misfolded proteins. These experiments demonstrate that the increase of uPA content in the yeast ER interferes with its folding. The data obtained in this work allow us to suggest that uPA is poorly secreted by yeast, because its folding requires milieu of the ER of higher eukaryotes. The unfavorable milieu in the yeast ER causes incorrect folding of significant portion of uPA, which is retained in the ER and undergoes either degradation or aggregation.
The H. polymorpha strains DL1-L (leu2) derived from DL-1 (ATCC 26012), 8 V (leu2) and 24–8 V (opu24 leu2) derived from CBS4732 (ATCC 34438) were described earlier [10, 16, 17]. The trp3 mutant DLT2 (leu2 mox, trp3::LEU2) was obtained by transformation of the DL1-L strain with the plasmid pMLTΔ, digested with Xho I and Ecl 136II as described earlier . The DL1-LΔM strain (leu2 Δmox) was obtained by transformation of DLT2 with the Xho I and Ecl 136II digested plasmid pSMΔ. The cassettes expressing different variants of uPA were introduced into the H. polymorpha genome either by replacement of the MOX gene , or via single copy integration of the expression vectors possessing the HpLEU2-d selectable marker into the LEU2 locus . The uPA expressing transformants are listed in Table 3. Integration into the MOX locus was performed via transformation of the DLT2 strain with the plasmids pSM1, pUR2SM1, pSMWC or pSMWCQ, digested with Xho I and Ecl 136II. Integration into the LEU2 locus was performed via transformation of the leu2 strains with the plasmids pAM226, pAM410, pNR4 or pNR5, digested with Ecl 136II. The S. cerevisiae strains expressing different uPA variants were obtained via integration of the Eco RV-digested expression vectors pNR9, pNR23, pNR26 and pNR27 into the URA3 locus of the YPH499 strain .
The plasmids used in this study are listed in Table 4. The uPA expression vectors pAM219, pAM226 and pAM281 were constructed by the insertion of sequence encoding uPA fused to the signal peptide of the Kluyveromyces lactis killer toxin  under control of the MOX promoter into the Bam HI and Hin dIII sites of the AMIpL1, AMIpLD1 and AMIpSL shuttle vectors , respectively. This insert consisted of a 1.8 kb Xho I-Bam HI fragment of the pSM1 plasmid  and an adapter, which was obtained by annealing the oligonucleotides 5'GATCCGCAGTCACACCAAGGAAGAGAATGGCCTGGCCCTCTGA3' and 5'AGCTTCAGAGGGCCAGGCCATTCTCTTCCTTGGTGTGACTGCG3'. To allow ligation of the Bam HI cohesive end of these vectors and the Xho I cohesive end of the insert, two bp of their overhangs were filled in by the Klenow enzyme. The expression vectors pAM410 and pSMWC encoding the N-terminally truncated uPA were constructed by insertion of a 0.8 kb Bgl I-Bam HI fragment of pWC28 (kindly given by M. Minashkin), carrying the sequence encoding uPA protease domain, into the Kpn I and Bam HI sites of the pAM226 and pSM1 plasmids, respectively (3'-terminal overhangs of Kpn I and Bgl I were removed by the Klenow enzyme). The plasmid pSMWCQ was constructed by replacement of the 0.7 kb Bam HI-Eco RI fragment of pSMWC for the corresponding fragment of pUR2SM . The expression vectors pNR4 and pNR5 encoding uPA variants with the KDEL ER retention signal were constructed by the insertion of an adapter, which was obtained by annealing the oligonucleotides 5'GATCAAGGACGAGCTGT3' and 5'AGCTACAGCTCGTCCTT3', between the Bam HI and Hin dIII sites of pAM226 and pAM410.
The S. cerevisiae uPA expression vectors were based on the YIpSEC-NR8 integrative shuttle vector, which was obtained from the YEpSEC1 plasmid  by deletion of the Sna BI-Aat II fragment, carrying the 2 μm DNA sequence and the LEU2 gene. The wild type uPA expression vector pNR9 was constructed by insertion of the 1.2 kb Kpn I-Hin dIII fragment of the pAM226 plasmid into the corresponding sites of the YIpSEC-NR8 plasmid. The uPA-C expression vector pNR23 was obtained by replacement of the Kpn I-Bam HI fragment in the pNR9 plasmid for the Bgl I-Bam HI fragment of the pWC28 plasmid (3'-terminal overhangs of Kpn I and Bgl I were removed by the Klenow enzyme). The uPA-Q and uPA-CQ expression vectors pNR26 and pNR27 were obtained by replacement of the Eco RI-Bam HI fragment in the plasmids pNR9 and pNR23 for the corresponding fragment of pUR2SM.
Culture conditions for the H. polymorpha strains and conditions for the induction of uPA expression in the CBS4732 derivatives were described previously . To induce uPA expression in transformants of the DL1-L strain, overnight YPD cultures were 6-fold diluted with YPM medium (2% Peptone, 1% Yeast extract, 2.5% methanol, 150 mM NaCl) and incubated at 37°C for 50–60 h.
Analyses of uPA expression
To prepare lysates, cells were disrupted with glass beads in TBST buffer (30 mM tris-HCl pH7.5, 150 mM NaCl, 2% Triton × 100), containing protease inhibitors, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM benzamidine, 0.1 mM sodium metabisulphite, 0.5 μg/ml TPCK, 0.5 μg/ml TLCK, 2.5 μg/ml antipain, 0.5 μg/ml leupeptine, 1.0 μg/ml pepstatin A, 2.0 μg/ml aprotinine. The protease inhibitors were omitted if lysates were analyzed for the uPA activity. Amounts of active uPA were determined by fibrinolytic activity assay . Total amounts of uPA in cell lysates and culture medium of different transformants were compared by probing appropriately diluted lysates and culture supernatants with the anti-uPA antibody specific to the uPA protease domain . The amounts of u-PA in culture medium were normalized to the levels of total cellular protein , whereas amounts of u-PA from cell lysates were normalized to the levels of soluble cellular protein in a sample assayed as described . To study uPA aggregation, cell debris was removed from lysates by centrifugation at 300 g for 10 min and lysates were centrifuged again at 10,000 g for 10 min. Obtained pellet and supernatant fractions were analyzed by Western blotting.
A qualitative test for the ability of yeast transformants to secrete uPA was performed by examination of their capacity to create haloes during growth on fibrin-containing media, as described previously [9, 18].
Transformation of H. polymorpha and Escherichia coli cells
This work was partially supported by the INTAS grant 01-0583.
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