FASE TRES: Estrategias Metacognitivas Una Herramienta Para Comprender Textos

3.2.1 In vivo protease resistance in the absence and presence of methotrexate

Protease treatment of DHFR had previously been shown to yield a stable fragment of roughly 25 kDa (Gaume et al., 1998). In order to determine the folding state of in vivo

expressed model substrates, their stability was tested upon protease treatment. For this purpose, isolated mitochondria from cells expressing the model substrates were solubilized and treated with proteinase K. The wild-type model substrates were partly degraded upon addition of the protease and a protease-resistant fragment of roughly 25 kDa was generated (Fig. 12, left panel, lane 2). In contrast, the mutant versions of the model substrates were entirely degraded (Fig. 12, right panel, lane 2) (Vestweber and Schatz, 1988). In presence of the DHFR substrate analog methotrexate, the wild-type model substrates were stabilized even further, and thus the levels of the stable fragment increased (Fig. 12, left panel, lane 3). This finding can be explained by binding of methotrexate to wild-type DHFR, which in turn leads to further stabilization of the native fold. Neither of the mutant forms was stabilized by methotrexate (Fig. 12, right panel, lane 3) because the substrate analog cannot bind to unfolded DHFR.

In conclusion, these results show that the wild-type DHFR constructs assume their native fold in the mitochondrial intermembrane space and in the matrix.

Figure 12. Folding of model substrates in the presence and absence of

methotrexate

Isolated mitochondria were solubilized with Triton X-100 and incubated with proteinase K (PK) for 20 min at 0 °C in the presence and absence of methotrexate (Mtx). Samples were analyzed by SDS-PAGE and immuno-staining with antibodies against DHFR. i, intermediate and m, mature form of IMS-DHFR. sf, stable fragment upon protease digestion.

3.2.2 Requirements for folding of DHFR in the IMS and matrix

Next, it was asked if nucleotides and heat stress have an effect on the folding state of the DHFR constructs. To this end, one set of isolated mitochondria from cells expressing the model substrates was depleted of nucleotides. The ATP levels of a second set of these mitochondria were kept high. Both sets were subjected to a short heat shock at 42 °C for 3 min. One set of samples was kept at 25 °C as a control. After solubilization of the mitochondria with Triton X-100, pellet and supernatant fractions, representing aggregated and soluble proteins, were separated by centrifugation. At 25 °C, IMS-DHFRWT _was

found in the soluble fraction in the presence and absence of ATP (Fig. 13 A, lanes 1-4). However, upon heat shock, the mature (m) form of IMS-DHFRWT aggregated in an ATP- dependent manner (Fig. 13 A, lanes 5-8). In the presence of ATP, 14 % of IMS-DHFRWT

aggregated (Fig. 13 A, lane 7). In the absence of ATP, aggregation increased to 89 % (Fig. 13 A, lane 5). The intermediate (i) form of IMS-DHFRWT_{, which is not yet cleaved}

by inner membrane peptidase and is thus still anchored to the inner membrane, aggregated almost completely upon heat shock (99 %) (Fig. 13 A, lanes 5+7).

Matrix-DHFRWT _{also aggregated in an ATP-dependent manner upon heat shock (Fig.}

13 B, lanes 5-7). However, only a much smaller proportion (1 % or 3 %) than in the intermembrane space aggregated in the matrix upon heat shock (Fig. 13 B, lanes 5+7). This could be attributed to a higher capacity of the protein quality control systems of the matrix than of the ones in the intermembrane space.

Figure 13. Aggregation of wild-type DHFR constructs in IMS and matrix

Isolated mitochondria were incubated under the indicated conditions, solubilized with Triton X-100, and soluble (S) and aggregate (P, pellet) fractions were separated by centrifugation and analyzed by SDS-PAGE and immuno-staining for indicated proteins. Singals of wild-type IMS-DHFR (A) and matrix-DHFR (B) were quantified in supernatant and pellet fractions and expressed as percentages of total protein. ND, not detectable. i, intermediate and m, mature form of IMS-DHFR.

RESULTS  

IMS-DHFRmut aggregated under all tested conditions (Fig. 14 A), even in the presence of ATP under physiological conditions (Fig. 14 A, lane 3). Matrix-DHFRmut behaved similar to matrix-DHFRWT _{(Fig. 14 B), although aggregation upon heat shock was slightly}

increased in comparison to the wild-type counterpart (Fig. 14 B, lanes 5+7). In the presence of ATP, only 10 % of matrix-DHFRWT _{aggregated upon heat shock (Fig. 4, lane}

7). This is in agreement with the ATP-dependent activity of the chaperones of the protein quality control system in the mitochondrial matrix.

Collectively, these results show that, under stress conditions, the stability of DHFR is determined by an ATP-dependent process. This applies to the wild-type constructs in the intermembrane space and in the matrix. Furthermore, it becomes clear that the capacity of the protein quality control system in the matrix is higher than the capacity of the protein quality control system in the intermembrane space. The unfolded DHFRmut construct was kept soluble in the matrix whereas its counterpart in the intermembrane space aggregated even under physiological conditions.

Figure 14. Aggregation of DHFRmut _{in IMS and matrix}

Isolated mitochondria were incubated under conditions to increase or decrease the mitochondrial ATP levels and then exposed to 25°C or 42°C for three minutes. Mitochondria were then solubilized with Triton X-100-containing buffer and soluble (S) and aggregate (P, pellet) fractions separated by centrifugation and analyzed by SDS- PAGE and immuno-staining using antibodies against the indicated proteins. The DHFR signals were quantified in the supernatant and pellet fractions and expressed as percentages of total protein. ND, not detectable. i, intermediate and m, mature form of IMS-DHFR.