Deciphering the molecular organization of GET pathway chaperones through native mass spectrometry

Get3/4/5 chaperone complex is responsible for targeting C-terminal tail-anchored membrane proteins to the endoplasmic reticulum. Despite the availability of several crystal structures of independent proteins and partial structures of subcomplexes, different models of oligomeric states and structural organization have been proposed for the protein complexes involved. Here, using native mass spectrometry (Native-MS), coupled with intact dissociation, we show that Get4/5 exclusively forms a tetramer using both Get5/5 and a novel Get4/4 dimerization interface. Addition of Get3 to this leads to a hexameric (Get3)(2)-(Get4)(2)-(Get5)(2) complex with closed-ring cyclic architecture. We further validate our claims through molecular modeling and mutational abrogation of the proposed interfaces. Native-MS has become a principal tool to determine the state of oligomeric organization of proteins. The work demonstrates that for multiprotein complexes, native-MS, coupled with molecular modeling and mutational perturbation, can provide an alternative route to render a detailed view of both the oligomeric states as well as the molecular interfaces involved. This is especially useful for large multiprotein complexes with large unstructured domains that make it recalcitrant to conventional structure determination approaches.

Automated summary

In this study, the authors used native mass spectrometry (Native-MS) to investigate the oligomeric states and structural organization of the Get3/4/5 chaperone complex. They discovered that Get4/5 forms a tetramer and interacts with Get4/4 through a novel dimerization interface. Addition of Get3 results in the formation of a hexameric complex with a closed-ring cyclic architecture. These findings provide molecular-level insights and demonstrate the importance of native-MS in studying large multiprotein complexes.