In situ structural analysis of Golgi intracisternal protein arrays

Significance To our knowledge, this is the first detailed study of Golgi ultrastructure within unperturbed cells. Three intracisternal structures were identified, with implications for Golgi architecture and trafficking: (i) Bundles of filaments show how cargoes may oligomerize to increase their local concentration at trans-Golgi buds. (ii) Granular aggregates provide evidence for cisternal maturation, as they are likely too large to transit the Golgi via vesicles. (iii) Protein arrays link the membranes of the central trans-Golgi cisternae, simultaneously maintaining the narrow luminal spacing while promoting cargo exit from the Golgi periphery by excluding material from the center. The asymmetry of the array structure indicates that the apposing membranes of a single cisterna have distinct compositions. The assembly of arrays may also enhance glycosyltransferase kinetics. We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo-electron tomography. These tomograms revealed structures within the Golgi cisternae that have not been seen before. Narrow trans-Golgi lumina were spanned by asymmetric membrane-associated protein arrays that had ∼6-nm lateral periodicity. Subtomogram averaging showed that the arrays may determine the narrow central spacing of the trans-Golgi cisternae through zipper-like interactions, thereby forcing cargo to the trans-Golgi periphery. Additionally, we observed dense granular aggregates within cisternae and intracisternal filament bundles associated with trans-Golgi buds. These native in situ structures provide new molecular insights into Golgi architecture and function.