Dual targeted mitochondrial proteins are characterized by lower MTS parameters and total net charge

Abstract

Background: In eukaryotic cells, identical proteins can be located in different subcellular compartments (termed dual-targeted proteins).

Methodology/principal findings: We divided a reference set of mitochondrial proteins (published single gene studies) into two groups: i) Dual targeted mitochondrial proteins and ii) Exclusive mitochondrial proteins. Mitochondrial proteins were considered dual-targeted if they were also found or predicted to be localized to the cytosol, the nucleus, the endoplasmic reticulum (ER) or the peroxisome. We found that dual localized mitochondrial proteins have i) A weaker mitochondrial targeting sequence (MitoProtII score, hydrophobic moment and number of basic residues) and ii) a lower whole-protein net charge, when compared to exclusive mitochondrial proteins. We have also generated an annotation list of dual-targeted proteins within the predicted yeast mitochondrial proteome. This considerably large group of dual-localized proteins comprises approximately one quarter of the predicted mitochondrial proteome. We supported this prediction by experimental verification of a subgroup of the predicted dual targeted proteins.

Conclusions/significance: Taken together, these results establish dual targeting as a widely abundant phenomenon that should affect our concepts of gene expression and protein function. Possible relationships between the MTS/mature sequence traits and protein dual targeting are discussed.

Figure 1. Dual localized proteins of the mitochondrial reference set are enriched for proteins with a low MitoProtII score.

Distribution of MitoProtII scores in dual localized (grey) and exclusive mitochondrial (white) proteins were analyzed using χ² test. Statistically significant differences in specific categories according to the χ² test (df = 1) are marked with asterisks (* p-value <0.05; ** p-value <0.005; *** p-value <0.001). Mitochondrial localization was determined according to the Mitop2 reference set.

Figure 2. Dual localized proteins of the mitochondrial reference set are enriched for proteins with a low total net charge.

Total Differences in the distribution of total net charge in dual localized (grey) and exclusively mitochondrial (white) proteins were analyzed using χ² test (p-value <0.001). Statistically significant differences in specific categories according to the χ² test (df = 1) are marked with asterisks (* p-value <0.05). Mitochondrial localization was determined according to the Mitop2 reference set.

Figure 3. α-complementation assay for mitochondrial and cytosolic location of predicted dual (A) and nondual (B) localized proteins.

Yeast expressing the indicated α fusion proteins and the ωc or ωm fragments were tested for color production on galactose medium, X-gal agar plates.

Figure 4. Proteins with a low net charge and low hydrophobic moment tend to be dual localized (more than exclusive mitochondrial proteins).

Differences in the distribution of total net charge in dual localized (black circles) and exclusively mitochondrial (white circles) proteins with either high (>6, top panel) or low (<6, bottom panel) hydrophobic moment were analyzed using χ² test. For proteins with a low hydrophobic moment (<6) there is a statistically significant difference between dual localized (black) and exclusively mitochondrial (white) proteins (p-value<0.02).