Evaluación asignaturas teórico-prácticas

5.2.1 PDEδ co-immunoprecipitates with Rac1 in ROS

To investigate if PDEδ and Rac1 interact in ROS, PDEδ and Rac1 co-immunoprecipitation assays from light- and dark-adapted ROS were performed. Light- and dark-adapted total ROS were solubilized with β-dodecylmaltoside. Rac1 was immunoprecipitated with anti-Rac1 antibodies, and the immunoprecipitates were subjected to Western blot analysis. As shown in Figure 34A, PDEδ co-immunoprecipitates with Rac1 in light-and dark-adapted ROS. More PDEδ in dark-adapted ROS indicates a stronger association of PDEδ with Rac1 in the dark- adapted state.

For further characterization of the light-regulated interaction of Rac1 and PDEδ both proteins were immunoprecipitated from the soluble and membranous fractions of light- and dark- adapted ROS. Immunoprecipitation of Rac1 from light- and dark-adapted soluble and membranous ROS fractions with anti-Rac1 revealed that consistent with the distribution of Rac1 in ROS (Figure 33A), the highest amount of Rac1 was immunoprecipitated from the membranous fractions (Figure 34B). In the soluble fractions less Rac1 was immunoprecipitated, also reflecting the situation in ROS as shown in Figure 33A. In all four fractions PDEδ was co-immunoprecipitated with Rac1, but in the soluble and membranous fractions of light-adapted ROS less PDEδ was co-immunoprecipitated. In the dark-adapted

Figure 33: Localization of Rac1 and PDEδ in light- and dark-adapted porcine retina. A) Western blots of total

lysates from light- and dark-adapted ROS (15µg) incubated with anti-Rac1 and anti-PDEδ antibodies. No significant difference in reactivity with these antibodies was detected, indicating that the amount of Rac1 and PDEδ remained unaltered in light- and dark-adapted ROS. B) light- and dark-adapted ROS corresponding to

15µg of protein (T) were lysed and fractionated into a soluble (S) and a membranous (M) fraction and separated by SDS-PAGE. Immunoblots with anti-Rac1 antibodies show that Rac1, primarily present in the membranous fractions, exhibited no significant differences in the distribution between the light- and dark-adapted states. Immunoblots with anti-PDEδ antibodies show that PDEδ was mainly detected in the soluble fractions, but was enriched in the membranes of dark adapted ROS. Western blot panels are representative of at least three independent experiments that gave the same result.

fractions more PDEδ was found in Rac1 immunoprecipitates, underlining that both proteins exhibit a stronger association in dark-adapted ROS (Figure 34B). In the dark-adapted soluble fraction of ROS the largest part of Rac1 was complexed with PDEδ. Here only a rather small amount of Rac1 is immunoprecipitated, nevertheless, compared to the amount of Rac1, the co-immunoprecipitated amount of PDEδ is very high. To confirm these result, we co- immunoprecipitated Rac1 from light- and dark-adapted soluble and membranous ROS fractions with anti-PDEδ antibodies (Figure 34C). The immunoprecipitation of PDEδ from light- and dark-adapted soluble and membranous ROS fractions also reflected the distribution of PDEδ in light- and dark-adapted ROS (Figure 33A), showing high amount of PDEδ in the soluble fractions of light- and dark-adapted ROS. PDEδ was enriched in the dark-adapted membranous fraction compared to the light-adapted membranous fraction, where PDEδ was present only in a very low amount. Co-immunoprecipitation of Rac1 occurred in the membranous fractions. In the light-adapted membranous fraction only very little Rac1 was detectable, whereas a higher amount of Rac1 co-immunoprecipitated with PDEδ in the dark- adapted membranous fraction (Figure 34C). These data support the notion that PDEδ and Rac1 exhibit a stronger association in dark-adapted ROS, and are there preferentially in the membranous fractions.

5.2.2 Isolation and analysis of native protein complexes from ROS by BN-PAGE

To further validate that PDEδ and Rac1 colocalize in ROS in native protein complexes, a two-dimensional approach, with native BN-PAGE as first dimension and denaturating SDS- PAGE as second dimension was applied (BN/SDS-PAGE). The use of non-ionic detergents allows the solubilization of native membrane protein complexes that can then be separated by

Figure 34: Light-dependent association

of Rac1 and PDEδ in ROS.

A) IPs from total lysates of light- and

dark-adapted ROS (500µg) using anti- Rac1 antibodies. Immunoprecipitated proteins were eluted with Laemi-buffer and the eluates were probed on Western blots with either anti-Rac1 or anti-PDEδ

antibodies. As shown in A, more PDEδ

coprecipitated with Rac1 in the dark- adapted state, indicating a stronger association of both proteins in the dark. B-C, equal amounts of protein (500µg), divided into soluble and membranous fractions, were immunoprecipitated using anti-Rac1 B) or anti-PDEδ

antibodies C), respectively. Eluates

were probed on Western blots with anti- Rac1 or anti-PDEδ. IP with anti-Rac1 antibodies showed that the highest amount of Rac1 was immunoprecipitated from the membranous fractions. In all four fractions PDEδ was co- immunoprecipitated with Rac1 but the highest amount of PDEδ was co- immunoprecipitated in the dark adapted fractions, indicating a stronger association of both proteins in dark- adapted ROS. Immunoprecipitates with anti-PDEδ antibodies showed the highest reactivity for PDEδ in the light- and dark-adapted soluble fractions and in the dark-adapted membranous fractions. Rac1 wasimmunoprecipitated in the light- and dark-adapted membranous fractions, but the strongest immunosignal for Rac1 was obtained in the dark adapted membranous fraction, again indicating a stronger association of both proteins in the dark. Immunosignals were absent from immunoprecipitates with normal mouse IgG (data not shown).

native electrophoretic methods ((Schagger et al., 1994), (Wittig and Schagger, 2005)). In the first dimension, solubilized native multiprotein complexes are separated according to their molecular masses. The second dimension resolves all individual components of the multiprotein complexes under denaturating conditions. All protein subunits released from one protein complex, are separated along the electric field gradient and are positioned in a straight line, one below the other, according to their molecular masses.

5.2.2.1 PDEδ and Rac1 colocalize in ROS in native protein complexes

Western blot analysis of the first dimension of BN-PAGE of soluble and membranous fractions of light- and dark-adapted ROS showed that PDEδ and Rac1 colocalize in dark- adapted ROS in the soluble and membranous fraction in a complex of app. 200 kDa (Figure 35, right panel). Because protein transfer from the first dimension onto blotting membranes is rather difficult and often inefficient, we also performed Western blot analysis of the second dimension of BN-PAGE of soluble and membranous fractions of light- and dark-adapted ROS.

Western blot analysis of the second dimension not only revealed that PDEδ and Rac1 colocalize in ROS but were found in diverse protein complexes depending on the light- (Figure 36A) or dark-adapted state (Figure 36B) of the retina. Colocalization of PDEδ and Rac1 was stronger in dark-adapted ROS, where both proteins colocalized in the soluble as well as in the membranous fraction. In dark-adapted ROS, PDEδ was part of distinct complexes ranging from high molecular weight complexes of 660 kDa to smaller complexes of around 90 kDa but the colocalization of PDEδ and Rac1 in the soluble and membranous fraction occurred in each case in different complexes (Figure 36B). In light-adapted ROS, no colocalization of PDEδ and Rac1 was detected in the soluble fraction. In the membranous fraction of light-adapted ROS, PDEδ and Rac1 colocalized in a single complex of approximately 300 kDa.

BN-PAGE revealed that PDEδ and Rac1 colocalize in light-and dark-adapted ROS and the stronger colocalization of both proteins in dark-adapted ROS support the data obtained from the co-immunoprecipitations (see Figure 34A-C), where both proteins also showed a stronger association in dark-adapted ROS.

Figure 35: Western blot of a first dimension of a BN-PAGE from light-and dark-

adapted ROS, soluble and membranous fractions. The BN lanes were excised from the gel and blotted onto PVDF membranes. Prior to electrotransfer using the semi-dry blotting method, the gel strips were incubated in a denaturating solution (1% SDS and 1% β-mercaptoethanol). After electrotransfer, the membrane strips were cut in the middle and one half was incubated with anti- Rac1 antibody and the other with anti-PDEδ antibody.