Related Experiment Videos
Creatine transporters: a reappraisal
Oliver Speer1, Lukas J Neukomm, Robyn M Murphy
1ETH-Zürich, Institute of Cell Biology, ETH-Hönggerberg, Zurich, Switzerland.
Molecular and Cellular Biochemistry
|February 24, 2004
Summary
Researchers developed antibodies to study creatine transporter (CrT) proteins. While initial results suggested mitochondrial localization, further analysis revealed cross-reactivity with other proteins, highlighting the need for caution in CrT research.
Area of Science:
- Cellular Metabolism
- Molecular Biology
- Biochemistry
Background:
- Creatine (Cr) is crucial for energy metabolism in cells like muscle and neurons.
- A high-affinity creatine transporter (CrT) facilitates cellular Cr uptake.
- Mutations in the creatine transporter gene (crt) cause severe developmental issues.
Purpose of the Study:
- To biochemically characterize creatine transporter (CrT) proteins.
- To raise and utilize antibodies against putative CrT-1 protein.
- To investigate CrT localization and function in cellular energy metabolism.
Main Methods:
- Antibodies were raised against synthetic peptides of the N- and C-terminal cDNA sequences of CrT-1.
- Western blotting was used to detect CrT polypeptides in tissue homogenates.
- Immunoelectron microscopy and subfractionation identified protein localization within mitochondria.
- Cr-uptake experiments with isolated mitochondria and plasma membrane vesicles were performed.
- Mass spectrometry identified immunologically reactive proteins.
Main Results:
- Anti-CrT antibodies recognized 70 and 55 kDa polypeptides in tissue homogenates.
- Mitochondrial Cr uptake was observed and could be blocked by anti-CrT antibodies.
- Mass spectrometry identified these reactive proteins as E2 components of alpha-keto acid dehydrogenase complexes, not CrT.
- Plasma membrane vesicles showed enrichment of a ~60 kDa polypeptide, likely the genuine plasma membrane CrT.
- Cross-reactivity of anti-CrT antibodies with non-CrT proteins was a significant finding.
Conclusions:
- Current anti-CrT antibodies cross-react with mitochondrial dehydrogenase E2 components, complicating biochemical characterization.
- A ~60 kDa polypeptide in skeletal muscle plasma membrane vesicles is a strong candidate for the genuine plasma membrane CrT.
- Mitochondria possess a low-affinity Cr uptake mechanism, but its biochemical identity remains elusive.
- Further research is essential to biochemically and biophysically characterize bona fide CrT polypeptides and mitochondrial transporters.
- Caution is advised when using existing anti-CrT antibodies for quantitation or studying CrT regulation.