, 2006) Without photoconversion, Dendra2 emits green fluorescenc

, 2006). Without photoconversion, Dendra2 emits green fluorescence. UV illumination

converts the pre-existing green fluorescent proteins to red so that they can be distinguished from newly synthesized proteins. We chased the degradation of SAX-3 by measuring the intensity Selleckchem DAPT change of red fluorescence at different time points postphotoconversion. At 7 hr after photoconversion, an average 35% of photoconverted SAX-3(WT)::Dendra was degraded in AVM at L1–L2 stages, and temperature rise had little effect on its degradation rate (Figures 6B and 6C). In contrast, over 50% of SAX-3(P37S)::Dendra was degraded at 20°C, and the level of degradation was further increased by temperature shift to 22.5°C (Figure 6C). In touch neurons, a fraction of SAX-3(P37S) was misfolded and either diffused or formed aggregates in the cytosol, whereas most SAX-3(WT) was in a native form and predominantly located on the cell surface (Figure 5C). Therefore, the difference of degradation rates between SAX-3(P37S) and SAX-3(WT) suggests that misfolded KU 55933 SAX-3 is overall more vulnerable to degradation than native SAX-3. Further, we found that the ebax-1(ju699) null mutation significantly reduced

the degradation of SAX-3(P37S) ( Figure 6D). Interestingly, a similar reduction was also observed in the degradation rate of SAX-3(WT) in ebax-1 mutants, suggesting that in vivo a pool of wild-type proteins,

possibly those in nonnative forms, relies on EBAX-1 for degradation. Supporting this finding, around 44% of AVM neurons in ebax-1 mutants showed aggregation of SAX-3(WT)-GFP, a 3-fold increase not over neurons in wild-type animals ( Figure S6E). Additionally, we found that after enriching misfolded proteins by proteasome inhibition in live worms, a fraction of SAX-3(WT) was detected in the EBAX-1 immunoprecipitant ( Figure S6F). In HEK293T cells expressing EBAX-1, ubiquitinated SAX-3(WT) was accumulated after proteasome activity was blocked for 4 hr ( Figure S6G). When the function of Hsp90 was further inhibited, the level of SAX-3 ubiquitination was increased ( Figure S6G), and SAX-3(WT) was recognized by EBAX-1 ( Figure S6H). Together, these data indicate that EBAX-1 can target misfolded wild-type SAX-3 as well as metastable mutant SAX-3. Consistent with the dependence of misfolded SAX-3 on EBAX-1 for degradation, we found that the AVM guidance defect in the sax-3(ky200) mutant showed strong sensitivity to the protein level of EBAX-1. Loss of endogenous EBAX-1 worsened the guidance defect caused by sax-3(ky200) at 20°C but did not further enhance the defect at 22.5°C. Overexpression of EBAX-1 significantly suppressed the guidance defect at 22.5°C ( Figure 7A).

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