Difference between revisions of "Part:BBa K1729002"

Latest revision as of 01:12, 22 October 2016

Yeast MRPS12 mitochondrial localization signal

This translational start sequence includes the Kozak and codon-optimized sequence for the mitochondrial localization signal of Saccharomyces cerevisiae mitochondrial ribosomal protein [http://www.yeastgenome.org/locus/S000005319/overview MRPS12]. The cleavage site was predicted based on identification of an R-10 motif (RxFxxTxxxx), which is typical of precursors cleaved sequentially by the yeast mitochondrial processing peptidase and mitochondrial intermediate peptidase (Branda et al.).

The part sequence corresponds to nucleotides 694840-694739 on chromosome XIV of the S. cerevisiae S288C reference genome. See also BBa_K1729001

References

Branda, Steven S et al. "Prediction and Identification of New Natural Substrates of the Yeast Mitochondrial Intermediate Peptidase." J Biol Chem 270 (1995): 27366-73.

Uses

Used in part BBa_K2077002. When connected to a fluorescent protein by a GS linker, mls targets fluorescence to yeast mitochondria even in strains with defunct mitochondria lacking ribosomal protein mRPS12, which is necessary for yeast mitochondria to translate their genome and make electron transport proteins needed to generate a membrane potential.

Figure 1: Observation of S. cerevisiae yeast cells that contain mls-yeGFP construct (test) with a 60X oil immersion objective. The right picture shows yeast cells under bright-field microscopy. The middle picture shows yeast cells under fluorescence microscopy. The left picture shows the overlay of the above mentioned two pictures.

Figure 2: Observation of S. cerevisiae yeast cells containing empty pSB416 GPD plasmid (negative control) with a 60X oil immersion objective. The right picture shows yeast cells under bright-field microscopy. The middle picture shows yeast cells under fluorescence microscopy. The left picture shows the overlay of the above mentioned two pictures.

Figure 3: Observation of S. cerevisiae yeast cells that contain mls-yeGFP construct (test) with a 60X oil immersion objective. The right picture shows yeast cells under bright-field microscopy. The middle picture shows yeast cells under fluorescence microscopy. The left picture shows the overlay of the above mentioned two pictures.

Figure 4: Comparison between Mito ID Red florescence, left, and mls-yeGFP florescence, right, in the mRPS12 TU knock out yeast strain.

Figure 5: Comparison between Mito ID Red florescence, left, and mls-yeGFP florescence, right, in the mRPS12 TU wild type yeast strain.

As shown in Figure 1 &3, scattered green fluorescence was observed inside some yeast cells. According to the pattern of fluorescence, we are able to conclude that GFP is targeted to some internal organelles rather than cytoplasm, and it is possible that the fluorescence comes from mitochondria. However, additional experiments with mitochondria fluorescence dye is needed to show the actual location of mitochondria in order to fully prove the function of mls. Comparing to Figure 1, the negative control (Figure 2) does not emit any fluorescence under the same excitation condition, indicating that empty pSB416 GPD plasmid did not affect mls-yeGFP expression. A blown-up image of fluorescing yeast cells is shown below in Figure 3. It clearly shows that the intensity of fluorescence varies throughout the cells, indicating mls protein is targeting GFP to certain regions of the cell, which possibly would be the mitochondria. The targeted fluorescence pattern of mls-yeGFP matches the fluorescence pattern of mitochondria targeting red fluorescent stain Mito ID Red, verifying that the mRPS12 mls preprotein peptide does target proteins to mitochondria (Figures 4 & 5). Additionally mRPS12 mls targets proteins to the mitochondria regardless of whether or not a yeast strain can translate its mitochondrial genome and/or use its electron transport chain for respiration, as illustrated by the lack of difference between fluorescence pattern similarities between the red stain and mls-yeGFP in both an mRPS12 TU, BBa_K1729001, knock out strain that has mitochondria that lack a functional electron transport chain and mitochondrial ribosomes, and the wild type strain with functional mitochondria (Figures 4 & 5).

Sequence and Features