Brain-type creatine kinase BB-CK interacts with the Golgi Matrix Protein GM130 in early prophase
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Creatine kinase (CK) isoenzymes are essential for storing, buffering and intracellular transport of “energy-rich” phosphate compounds in tissues with fluctuating high energy demand such as muscle, brain and other tissues and cells where CK is expressed. In brain and many non-muscle cells, ubiquitous cytosolic “brain-type” BB-CK and ubiquitous mitochondrial CK (uMtCK) act as components of a phosphocreatine shuttle to maintain cellular energy pools and distribute energy flux. To date, still relatively little is known about direct coupling of functional dimeric BB-CK with other partner proteins or enzymes that are important for cell function. Using a global yeast two-hybrid (Y2H) screen with monomeric B-CK as bait and a representative brain cDNA library to search for interaction partners of B-CK with proteins of the brain, we repeatedly identified the cis-Golgi Matrix protein (GM130) as recurrent interacting partner of B-CK. Since HeLa cells also express both BB-CK and GM130, we subsequently used this cellular model system to verify and characterize the BB-CK-GM130 complex by GST-pulldown experiments, as well as by in vivo co-localization studies with confocal microscopy. Using dividing HeLa cells, we report here for the first time that GM130 and BB-CK co-localize specifically in a transient fashion during early prophase of mitosis, when GM130 plays an important role in Golgi fragmentation that starts also at early prophase. These data may shed new light on BB-CK function for energy provision for Golgi-fragmentation that is initiated by cell signalling cascades in the early phases of mitosis.
Keywordscreatine kinase energy metabolism Golgi Matrix protein GM130 mitosis yeast two-hybrid energy shuttle Mayven
refers to either the ubiquitous brain-type CK subunit, B-CK cDNA constructs or B-CK bait constructs, whereas BB-CK refers to the functional cytosolic, ubiquitous brain-type CK dimer complex found in vivo
ubiquitous mitochondrial CK
Glutathione-S-transferase and Y2H refers to yeast two-hybrid
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We kindly thank Dr. Francis Barr for generously providing the rat GM130 constructs and Dr. Jean-Paul Steghens for providing the human B-CK cDNA; Tina Thurnherr and Dr. Elisabeth Ehler for critical reading of the manuscript. Uwe Schlattner is acknowledged for advice for the design of the B-CK bait construct and for supervision during the initial phase of this work. We also thank Dr. Ulricke Kutay and the members of our group for helpful discussions. This work was supported by a Swiss National Foundation Grant No. 3100A0–102075.
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