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. 2011 Mar 1;7(3):346–350. doi: 10.4161/auto.7.3.14234

Figure 1.

Figure 1

Modulation of Alfy levels permits starvation-mediated macroautophagy, but modulates the degree of proteolysis in response to starvation. (A) Long-lived protein degradation (LLPD) upon depletion of Alfy by siRNA. HeLa cells were transfected with a scramble siRN A sequence (siCtrl) or a sequence against Alfy (siAlfy). 48 h later, long-lived proteins were labeled with [14C]-valine. Cells were placed in complete media (CM), Hank's Basic Salt Solution (HBSS) with 10 mM HEPES (Starve) or HBSS with 10 mM HEPES plus 10 mM 3-methyladenine (Starve+3MA). Under all conditions 10 mM cold valine was also supplemented. After 4 to 6 h, cells were collected and processed by TCA precipitation and measured for proteolysis as previously described in reference 20. ANOVA revealed that upon Alfy depletion there was a significant increase of proteolysis under starvation conditions (Starve, F(1,22) = 5.430; *p = 0.0294). There was no significant difference under nonstarved (CM, F(1,22) = 0.101; p = 0.9782) or starvation + 3MA treated (Starve+3MA, F(1,6) = 1.999; p = 0.2071) conditions (N.S. = ‘not significant’). In both siCtrl and siAlfy transfected cells, starvation led to a significant increase in % proteolysis (siCtrl, p = 0.0207; siAlfy, p = 0.0001) but not in the presence of 3MA (siCtrl, p = 0.8878; siAlfy, p = 0.45). (B) LLPD upon overexpression of Alfy. HeLa cells were transfected with an empty vector (Mock) or a vector encoding the p62- and Atg5-binding C terminus of Alfy (amino acids 2,285–3,526) that was previously shown to increase aggregate-clearance (Alfy C).20 LLPD was monitored as described in (A). ANOVA revealed that overexpression of Alfy leads to significantly less proteolysis upon starvation (Starve, F(1,4) = 8.047; *p = 0.0470). There was no significant difference under unstarved (CM, F(1,4) = 0.115; p = 0.7511) or starvation + 3MA treated (Starve+3MA, F(1,6) = 0.510; p = 0.5145) conditions. In both Mock and Alfy C transfected cells, starvation led to a significant increase in % proteolysis (Mock, p = 0.0001; Alfy C, p = 0.0221) which was significantly inhibited by 3MA (Mock, p = 0.001; Alfy C, p = 0.0473).