UCP3 expressed in yeast is primarily localized in extramitochondrial particles

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Uncoupling protein 1 (UCP1) is well-characterized for its role in thermogenesis.
• Previous studies suggested UCP3 and UCP3s exhibit a deranged state upon expression in Saccharomyces cerevisiae, leading to unregulated uncoupling.

Purpose of the Study:

• To investigate the localization and aggregation state of UCP3 and UCP3s when expressed in Saccharomyces cerevisiae.
• To elucidate the reasons behind the observed uncoupling effects of UCP3 and UCP3s.

Main Methods:

• Expression of UCP3 and UCP3s in Saccharomyces cerevisiae using varying expression vectors (medium, high, very high).
• Solubility assays using nonionic detergents (Triton X100) to differentiate UCP3/UCP3s from UCP1.
• Fractionation and visual inspection of cellular components to identify extramitochondrial aggregates.

Main Results:

• The majority of UCP3 and UCP3s were found in extramitochondrial aggregates, irrespective of expression levels.
• UCP3 and UCP3s showed insolubility in Triton X100, unlike UCP1.
• Very high expression led to visible extramitochondrial particles and inclusion body-like aggregates within the mitochondrial fraction.

Conclusions:

• The bulk of expressed UCP3 and UCP3s forms extramitochondrial aggregates.
• The presence of small amounts of incorporated, dysfunctional UCP3/UCP3s inhibits the incorporation of the remaining protein into mitochondria.
• This aggregation behavior offers a novel assay for assessing the state of heterologously expressed mitochondrial membrane proteins.