Team:SCAU-China/Proof Of Concept

MESEG

Proof Of Concept

As a proof of concept of MESEG, we expressed the fusion protein (SpMTL-LEAAAK-CC-mCh-ARR ) in yeast and tested its function. The figure bellow is a brief introduction of the structure of SpMTL-LEAAAK-CC-mCh-ARR.

Figure 1. Structure of SpMTL-LEAAAK-CC-mCh-ARR. In yeast, the receptor ATG19 is required for the vacuolar deposition of Ape1, a vacuolar hydrolase. It binds Ape1 through its Ams1 binding domain (ABD) in the middle and binds ATG8 through the ARR domain at its C-terminal (Yamasaki and Noda, 2017). It also can self-oligomerize through its coiled-coil (CC) region. We kept the ARR domain and CC region, but replaced the ABD with mCherry florescence protein (mCh). At the N-terminal of fusion protein, a Cd binding protein derived from Sedum plumbizincicola was added. To prevent the individual structural domains from interfering with each other, we also added a linker (EAAAK) between the CC region and SpMTL.

We first tested the ability of heavy metal scavenging of the fusion protein. When expressed in the Cd-sensitive mutant Δycf1, the fusion protein restored its Cd tolerance to a level comparable to that of yeast wild type (Figure 2). Moreover, using confocal laser scanning microscopy, we found that the fusion protein SpMTL-LEAAAK-CC-mCh-ARR is recruited to pre-autophagosomal structure (PAS) under nitrogen starvation conditions, which is quite distinct from the cytosolic protein SpMTL-mCherry (Figure 3). The subcellular localization of SpMTL-LEAAAK-CC-mCh-ARR is similar to that of autophagic marker ATG8, implying that that it might behave like cargo receptor ATG19 and is transported to the vacuole via autophagy route. These results indicate that the fusion protein SpMTL-LEAAAK-CC-mCh-ARR might function as a molecular megnet for heavy metal scavenging.

Figure 2. Expressing the fusion protein SpMTL-LEAAAK-CC-mCh-ARR restored the Cd tolerance of yeast mutant Δycf1.


The wild-type yeast strain BY4741 or Cd-sensitive mutant Δycf1 were transformed with EV (pYES2 empty vector), or pYES2 carrying SpMTL-LEAAAK-CC-mCh-ARR, and grown on SD plates with indicated concentrations of CdCl2 for 3 d.

Figure 3: The subcellular localizations of fusion proteins SpMTL-mCherry and SpMTL-LEAAAK-CC-mCh-ARR

The Δycf1 mutant were transformed with vectors expressing SpMTL-mCherry or SpMTL-LEAAAK-CC-mCh-ARR fusion proteins, and grown under nitrogen deficiency conditions for 12 hr before Confocal microscopy observation.V.vacuole.




  1. Yamasaki, A., and Noda, N.N. (2017). Structural Biology of the Cvt Pathway. J MOL BIOL 429, 531-542.