Association of calcineurin with the COPI protein Sec28 and the COPII protein Sec13 revealed by quantitative proteomics

Abstract

Calcineurin is a calcium-calmodulin-dependent serine/threonine specific protein phosphatase operating in key cellular processes governing responses to extracellular cues. Calcineurin is essential for growth at high temperature and virulence of the human fungal pathogen Cryptococcus neoformans but the underlying mechanism is unknown. We performed a mass spectrometry analysis to identify proteins that associate with the calcineurin A catalytic subunit (Cna1) in C. neoformans cells grown under non-stress and high temperature stress conditions. A novel prioritization strategy for mass spectrometry data from immunoprecipitation experiments identified putative substrates and proteins potentially operating with calcineurin in common pathways. Cna1 co-purified with proteins involved in membrane trafficking including the COPI component Sec28 and the COPII component Sec13. The association of Cna1 with Sec28 and Sec13 was confirmed by co-immunoprecipitation. Cna1 exhibited a dramatic change in subcellular localization during high temperature stress from diffuse cytoplasmic to ER-associated puncta and the mother-bud neck and co-localized with Sec28 and Sec13.

Figure 1. Mass spectrometry (MS) analysis of calcineurin-associated proteins.

A. A Coomassie blue-stained gel shows that the GFP-Trap resin exhibits a very low affinity towards proteins present in the control lysate, whereas GFP-Cna1 (star) associates with a large number of proteins. An arrow indicates a protein that likely corresponds to FKBP12 in a sample treated with FK506. B. Scheme depicting general proteomics approach/strategy. C. Coomassie blue-stained gels of the samples that were precipitated using the GFP-Trap resin (left) and the cell lysates analyzed for the protein content as a control (right). For each gel, an area that was excised from the gel for the subsequent MS analysis is depicted (brackets).

Figure 2. Scheme illustrating the strategy to identify calcineurin-interacting proteins.

In principle, a protein that was identified in the immunoprecipitated sample from GFP-Cna1-expressing strain but not from control strain, and which was not detected in the lysate was considered a high affinity interactant. Conversely, a protein that was highly abundant in the lysate and not detected in the purified sample from GFP-Cna1-expressing strain was categorized as non-interacting with Cna1. Other categories reflect the relative amounts of proteins detected in lysates and immunoprecipitated samples.

Figure 3. Sec13 and Sec28 co-precipitate with calcineurin A from cell lysates.

A. A co-IP of the GFP-Sec13 with the mCherry-Cna1 using the RFP-Trap resin. A strain that expresses only GFP-Sec13 and a strain that expresses only mCherry-Cna1 served as negative controls. The membrane was initially probed with an anti-GFP antibody, stripped and subsequently probed with an anti-dsRed antibody to detect the precipitated mCherry-Cna1. B. A co-IP of GFP-Sec28 with mCherry-Cna1 using the RFP-Trap resin. A strain that expresses only GFP-Sec28 and a strain that expresses mCherry-Cna1 but not GFP-Sec28 served as negative controls. The membrane was probed with an anti-GFP antibody and subsequently probed with an anti-dsRed antibody to detect the precipitated mCherry-Cna1.

Figure 4. mCherry-Cna1 co-localizes with (A) GFP-Sec13 and with (B) GFP-Sec28 during a rapid temperature shift to 37°C.

Cells were initially grown at 24°C, and the slide containing the cells grown on an agarose patch was placed on a heating block preset to 37°C. The cells were imaged after 30 minutes of incubation at 37°C. Scale bar equals 5 µm.

Figure 5. A hypothetical model showing multiple processes controlled by calcineurin during thermal stress in C. neoformans.

The model is based on findings presented in this study (the association of calcineurin catalytic subunit (Cna1) with trehalose synthase Tps1, Slm1, and ER chaperones, a putative role of Cna1 in vacuole morphology, and localization of Cna1 to the ER during thermal stress), our other studies (co-localization of Cna1 with ER-associated sites of mRNA processing, P-bodies and stress granules during stress), and the work of others.