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Forschungsarbeit

Hep1 – a chaperone for a chaperone

Von Dr. Marta Blamowska (19.02.2014)

Proteins are the most versatile and complex biomolecules, playing an active role in the vast majority of biological processes. Although around 20 % of eukaryotic proteins remain intrinsically disordered in vivo, a great number of proteins need to adopt a proper three-dimensional structure, which is critical for their physiological function. Folding process of the majority of proteins requires assistance of highly specialized folding helpers called molecular chaperones. One of the main classes of molecular chaperones is the Hsp70 chaperone class. 

Chaperones of this class are essential for a number of cellular processes and are present in the majority of cellular compartments, such as the cytosol, the nucleus, the endoplasmic reticulum, the chloroplasts and the mitochondria. The mitochondrial Hsp70 protein, mtHsp70, resides in the mitochondrial matrix where it fulfils a number of chaperone functions, including protein folding and prevention of protein aggregation. Most importantly, however, as an essential component of the import motor of the TIM23 translocase, mtHsp70 drives the ATP-dependent translocation of proteins into the mitochondrial matrix. 

Surprisingly, mtHsp70 itself has a propensity to self-aggregate. Therefore, it requires the Hsp70 escort protein Hep1, which prevents mtHsp70 aggregation and keeps mtHsp70 in a functional state. However, the molecular basis for the propensity to aggregate of mtHsp70 as well as for the interaction between mtHsp70 and its escort protein has not been understood.

Within the first two years of my PhD project I have characterized the structural determinants of mtHsp70 that make it prone to aggregation and analysed the structural requirements of mtHsp70 for its interaction with the escort protein Hep1.

Blamowska: Abb. 1[Bildunterschrift / Subline]: Fig.: Model of the folding pathway of mtHsp70. The ATPase domain (blue) and the PBD (pink) of mtHsp70 fold independently. The ATPase domain undergoes a Hep1-dependent and an ATP/ADP-dependent step in the folding process. The PBD domain folds spontaneously and, for simplicity, it is depicted in a folded state in all folding intermediates of mtHsp70, although it presumably folds simultaneously with the ATPase domain. In the final folding step, both domains cooperate together to acquire the final structure of mtHsp70. The presented model depicts additionally a transition of native mtHsp70 to the aggregation-prone conformer and the anti-aggregation action of Hep1 (green).

The aggregation studies demonstrate that a variant of Ssc1 consisting of the ATPase domain and the subsequent interdomain linker aggregates in absence of Hep1. In contrast, the peptide binding domain (PBD) and the ATPase domain alone are not prone to aggregation. Moreover, the interaction studies reveal that the aggregation-prone region seems to be the smallest entity within Ssc1 required for the interaction with Hep1. Taken together, the native Ssc1 adopts an aggregation-prone conformation, in which the ATPase domain with the interdomain linker has the propensity to aggregate. Hep1 binds to this aggregation-prone region and thereby counteracts the aggregation process and keeps the native Ssc1 in a functional and active state.

Although Hsp70 chaperones are important for the biogenesis of a multitude of proteins, little is known about the biogenesis of these chaperones themselves. In the last two years of the PhD project, I analyzed the de novo folding process of the mtHsp70 chaperone. To this end, I established in organello, in vivo and in vitro assays that were then employed to study the de novo folding of Ssc1. 

Upon import into mitochondria, Ssc1 folds rapidly with the ATPase domain and the PBD adopting their structures independently of each other. Notably, the ATPase domain requires the presence of the interdomain linker for its folding, whereas the PBD folds without the linker. Moreover, in the absence of Hep1, the ATPase domain with the interdomain linker displays a severe folding defect, which indicates a role of Hep1 in the folding process of Ssc1. Apart from Hep1, none of the general mitochondrial chaperone systems seems to be important for the folding of Ssc1. Furthermore, the folding process of Ssc1 was reconstituted in vitro and the main steps of the folding pathway of Ssc1 were characterised. Hep1 and ATP/ADP are required and sufficient for the folding of Ssc1 into the native, catalytically active form. In an early step of folding, Hep1 interacts with the folding intermediate of Ssc1. This interaction induces conformational changes which allow binding of ATP/ADP. The binding of a nucleotide triggers Hep1 release and further folding of the intermediate into a native Ssc1. This study provided the first direct evidence for the requirement of Hep1 for the folding of the Ssc1 chaperone. Thus, it demonstrated for the first time that the de novo folding of an Hsp70 chaperone depends on a specialized proteinaceous factor. 

In conclusion, Hep1 fulfils a dual chaperone function in the cell. It mediates the de novo folding of Ssc1 and maintains folded Ssc1 in a functional state during the ATPase cycle. Therefore, the Hep1 chaperone plays a crucial role for the protein biogenesis and homeostasis in mitochondria.


Scientific career
  • 2002 - 2007
  • Master course in Biotechnology, specialization in Biochemistry, Jagiellonian University of Cracow, Poland
  • 2008 - 2012
  • PhD project, Adolf-Butenandt Institute, Physiological Chemistry, LMU München
  • 2013
  • Advanced Training to Clinical Research Associate, Pharmaakademie Bremen

Publications
  • * Schusdziarra, C., Blamowska, M., Azem, A., Hell, K., Methylation-controlled J-protein MCJ acts in the import of proteins into human mitochondria. Human Molecular Genetics, 2013. Apr 1; 22(7): pp. 1348-1357
  • * Blamowska, M., Neupert, W., Hell, K., Biogenesis of the mitochondrial Hsp70 chaperone. Journal of Cell Biology, 2012. Oct 1;199(1): pp. 125-135
  • * Blamowska, M., Sichting, M., Mapa, K., Mokraniac, D., Neupert, W., Hell, K., ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1. Journal of Biological Chemistry, 2009. 285(7): pp. 4423-4431