The biogenesis of multispanning membrane proteins at the Sec61-translocon and their integration into the lipid bilayer of the endoplasmic reticulum

The key entry point of most membrane proteins into the lipid bilayer is the Sec61/SecYEG translocon, that mediates the transfer of hydrophilic sequences across the membrane and integration of mostly apolar a-helical transmembrane domains into the lipid bilayer. Three distinct integrations steps can be distinguished: (1) a first hydrophobic signal sequence targets the protein to the translocon, integrates itself into the membrane, and initiates translocation of the downstream polypeptide. (2) A subsequent hydrophobic segment laterally exits the translocon into the bilayer and thus stops further transfer. (3) The next hydrophobic sequence triggers re-integration into the translocon, re-initiating polypeptide transfer. Successive stop-transfer and reintegration sequences result in complex multispanning proteins. The major determinant of membrane topology appears to be the hydrophobicity of transmembrane domains. This has been best demonstrated for potential stop-transfer segments, suggesting a sequence-autonomous thermodynamic equilibration between the hydrophilic environment of the translocon and the apolar lipid phase.In this thesis, we analyzed in detail the hydrophobicity threshold for a potential re-integration TM domain downstream of different cytoplasmic loop sequences. Surprisingly, we discovered a strong dependence on the length of this cytoplasmic sequence. Short sequences are facilitating re-integration, while long ones seem to impede it. This demonstrates, that re-integration is not independent from the sequence-context. Further investigations revealed that loop sequences containing isolated folding domains, intrinsically disordered sequences, or sequences with a high affinity for chaperones enhance the reintegration efficiency, whereas those with low affinity to chaperones, and fragments of natural protein domains impair re-integration. We propose that the latter sequences, as they collapse to molten globules – i.e. near-native conformation of high compactness with already pronounced secondary structure and increased amount of hydrophobic residues on the surface area – compete with the translocon for interaction with the potential transmembrane segment. Our results thus define the environment of the nascent polypeptide chain when re-integration can occur and may serve as a guide in de novo membrane protein design. In a second part, we characterized the antiviral natural product cavinafungin as an inhibitor of signal peptidase for Dengue virus as well as host substrates, inhibiting biogenesis of viral proteins from a single precursor membrane polyprotein

Membrane Protein Integration and Topogenesis at the ER

Most membrane proteins are composed of hydrophobic α-helical transmembrane segments and are integrated into the lipid bilayer of the endoplasmic reticulum by the highly conserved Sec61 translocon. With respect to the integration mechanism, three types of transmembrane segments can be distinguished-the signal, the stop-transfer sequence, and the re-integration sequence-which in linear succession can account for all kinds of membrane protein topologies. The transmembrane orientation of the initial signal and to a weaker extent also of downstream transmembrane segments is affected by charged flanking residues according to the so-called positive-inside rule. The main driving force for transmembrane integration is hydrophobicity. Systematic analysis suggested thermodynamic equilibration of each peptide segment in the translocon with the membrane as the underlying mechanism. However, there is evidence that integration is not entirely sequence-autonomous, but depends also on the sequence context, from very closely spaced transmembrane segments to the folding state and properties of neighboring sequences. Topogenesis is even influenced by accessory proteins that appear to act as intramembrane chaperones

Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration

The Natural Product Cavinafungin Selectively Interferes with Zika and Dengue Virus Replication by Inhibition of the Host Signal Peptidase

Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses