Autophagic flux is required for the synthesis of triacylglycerols and ribosomal protein turnover in Chlamydomonas

Abstract

Autophagy is an intracellular catabolic process that allows cells to recycle unneeded or damaged material to maintain cellular homeostasis. This highly dynamic process is characterized by the formation of double-membrane vesicles called autophagosomes, which engulf and deliver the cargo to the vacuole. Flow of material through the autophagy pathway and its degradation in the vacuole is known as autophagic flux, and reflects the autophagic degradation activity. A number of assays have been developed to determine autophagic flux in yeasts, mammals, and plants, but it has not been examined yet in algae. Here we analyzed autophagic flux in the model green alga Chlamydomonas reinhardtii. By monitoring specific autophagy markers such as ATG8 lipidation and using immunofluorescence and electron microscopy techniques, we show that concanamycin A, a vacuolar ATPase inhibitor, blocks autophagic flux in Chlamydomonas. Our results revealed that vacuolar lytic function is needed for the synthesis of triacylglycerols and the formation of lipid bodies in nitrogen- or phosphate-starved cells. Moreover, we found that concanamycin A treatment prevented the degradation of ribosomal proteins RPS6 and RPL37 under nitrogen or phosphate deprivation. These results indicate that autophagy might play an important role in the regulation of lipid metabolism and the recycling of ribosomal proteins under nutrient limitation in Chlamydomonas.

Fig. 1.

Concanamycin A (ConcA) treatment results in ATG8 accumulation in Chlamydomonas cells. (A) Chlamydomonas cells in exponential growth phase were treated with increasing concentrations (0, 0.10, 0.20, and 0.40 µM) of ConcA for 12 h. (B) Chlamydomonas cells in exponential growth phase were treated with 0.1 µM ConcA for 12 h. Samples of non-treated cells were taken as the same time as a control. A 20 µg aliquot of total extracts from ConcA-treated cells was incubated in the absence (–) or presence (+) of 500 U ml^–1 phospholipase D (PLD) at 37 °C for 3 h. (C) Chlamydomonas cells in exponential growth phase were treated with 0.1 µM ConcA for various times (1, 2, 4, 8, and 24 h). Samples of non-treated cells were taken at the initial and the latest time (0 and 24 h, respectively) and used as control. For (A), (B), and (C) 20 µg of total extracts were resolved by 15 % SDS–PAGE followed by western blotting with anti-ATG8 and anti-FKBP12 antibodies. The lipidated form of ATG8 (ATG8-PE) is indicated. Molecular mass markers (kDa) are indicated on the left. (D) Immunolocalization of ATG8 in Chlamydomonas cells treated with 0.1 µM ConcA. Chlamydomonas cells growing exponentially were treated with 0.1 μM ConcA for 2, 4, or 8 h. Non-treated cells at 8 h were used as control. Cells were collected and processed for immunofluorescence microscopy analysis with anti-ATG8 antibodies. Scale bar=8 μm.

Fig. 2.

Ultrastructural analysis of Chlamydomonas cells treated with concanamycin A (ConcA). Electron microscopy images from Chlamydomonas cells treated with 0.1 µM ConcA for 0 h (control cells, A), 4 h (B), or 8 h (C). Enlargement of (A), (B), and (C) showing vacuoles of untreated cells (D), ConcA-treated cells for 4 h (E), and 8 h (F and G). v, vacuole. Scale bars=2 µm (A), 1 µm (B, C), 500 nm (D–G).

Fig. 3.

Inhibition of autophagic flux by concanamycin A (ConcA) prevents the degradation of ribosomal proteins under nitrogen starvation or phosphate limitation. (A) Chlamydomonas cells growing exponentially in Tris-acetate phosphate medium (TAP) were washed twice with nitrogen-free medium (TAP-N) and grown under these conditions for 4, 8, and 24 h. Control cells were washed in TAP medium and grown in the presence of nitrogen. (B) Chlamydomonas cells growing in TAP medium were washed with a nitrogen-free medium and grown under these conditions for 16 h in the absence (–) or presence (+) of 0.1 µM ConcA. (C) Chlamydomonas cells growing exponentially in TAP medium were washed with a phosphate-free medium (TA) and grown under these conditions during 8, 24, and 48 h. Control cells were washed and resuspended in TAP medium. (D) Chlamydomonas cells growing in TAP medium were washed with a phosphate-free (TA) medium and grown under these conditions for 48 h. Before collecting samples, cells were treated for 24 h with 0.1 µM ConcA. For (A–D), 20 µg of total extracts were resolved by 12% (RPS6) or 15% (RPL37, ATG8, and FKBP12) SDS–PAGE followed by western blotting with anti-OLLAS, anti-RPL37, anti-ATG8, and anti-FKBP12 antibodies. Molecular mass markers (kDa) are indicated on the left.

Fig. 4.

Concanamycin A (ConcA) prevents the formation of lipid bodies and the synthesis of TAGs in nitrogen- or phosphate-limited cells. (A) Chlamydomonas cells growing exponentially in TAP medium were treated as described in Fig. 3B and D for nitrogen or phosphate limitation, respectively, in the absence or presence of 0.1 µM ConcA. Lipid bodies were stained with Nile red and imaged by fluorescence microscopy. Scale bar=8 µm. (B) Lipid bodies from Chlamydomonas cells growing under the same conditions as described in (A) were stained with Nile red and the corresponding fluorescence was analyzed and quantified by flow cytometry (see the Materials and methods). (C) Quantification of triacylglycerols (TAGs) from Chlamydomonas cells subjected to nitrogen or phosphate limitation in the presence of 0.1 µM ConcA. Four biological replicates were analyzed for each condition. **Differences were significant at P<0.001 according to the Student’s t-test. *P<0.05.