Arad, S. | Search bioRxiv

bioRxiv2025

Structural basis of an EMC:Spf1 insertase-dislocase complex in the eukaryotic endoplasmic reticulum

Most eukaryotic membrane proteins are inserted into the membrane at the endoplasmic reticulum (ER). This essential but error-prone process relies on molecular quality control machineries to prevent mistargeting and incorrect structure formation. Here we show that the ER membrane protein complex (EMC) forms an evolutionarily conserved supercomplex with the P5A-ATPase Spf1/ATP13A1. This supercomplex combines the transmembrane domain (TMD) insertase function of the EMC and the TMD dislocase activity of Spf1 in a single entity. Our cryo-EM structure of this supercomplex shows that EMC and Spf1 form a shared intramembrane cavity for substrate engagement and reveals that the ATPase cycle of Spf1 regulates access to this cavity. Together, our study suggests that proteins with opposing biochemical activities in membrane protein biogenesis - insertion versus dislocation - form an integrated molecular machine in eukaryotes to proofread membrane protein insertion and topogenesis.

biochemistry↗

bioRxiv2024

A new targeting motif for endoplasmic reticulum surface proteins

The Endoplasmic Reticulum (ER) is the entry site to the secretory pathway, serving as the targeting destination for [~]30% of the proteome. The mechanisms for targeting soluble or integral membrane secretory pathway proteins are well-studied. However, it is currently unknown how the tens of ER surface proteins (SuPs), central for organelle function, reach the outer leaflet of the membrane. It was previously shown that an amphipathic helix (AH) from the Brome mosaic virus protein 1a, is both necessary and sufficient for targeting to the ER surface in bakers yeast. We therefore utilized this helix as a model substrate and performed a high-content screen to uncover factors that affect SuP targeting. Our results suggest a role for membrane lipid composition in targeting specificity. To see if the presence of an AH is a more general mechanism for SuP targeting, we searched for their presence within SuPs of both yeast and humans. Five endogenous yeast SuPs contained AHs, and of these four were sufficient for ER localization. Moreover, the presence of an AH was conserved to human SuP orthologs. By altering helix features we determine the parameters that affect this new targeting motif. Hence our work demonstrates how specific properties of AHs encode affinity for the ER membrane. More globally, understanding how SuPs are targeted correctly takes us a step forward in determining the underlying mechanisms of cellular localization and secretory pathway functions. The authors declare that they have no conflict of interest.

cell biology↗