Folding states of transit peptides and mature peptides in the cytosol: implications for the import-competent state of preproteins.

Nucleotide-binding motifs nucleated folding of the first enzymes

Presequences of non-imported mitochondrial proteins serve as quality control elements in the cytosol

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with presequences which serve as targeting signals for the mitochondrial matrix, where they are cleaved by the mitochondrial processing peptidase (MPP). In this study, we comprehensively elucidated the role of the presequence and the mature part of mitochondrial precursors in the cytosol, by use of a cytosol-targeted MPP which prematurely processed mitochondrial precursors. Over time, cytoMPP resulted in mitochondrial depletion. However, the cellular response to cytoMPP was surprisingly different to that observed for other models of mitochondrial import inhibition. Cytosolic maturation rendered many proteins stable in the cytosol, indicating that their mature parts lack ubiquitination signals. Accordingly, cytoMPP did not induce the upregulation of the proteasome, which normally is a hallmark of mitochondrial dysfunction. Instead, cytoMPP elicited a heat shock response and impaired the sequestration of precursors in the cytosol. Our observations demonstrate that mitochondrial presequences are more than just address labels. Rather, they play an important role in quality control and orchestrate the cellular response to defects in mitochondrial protein import.

of the native Sec61 protein-conducting channel

Translocon Remodeling Modulates Ribosomal Frameshifting and the Maturation of the Sindbis Virus Structural Polyprotein

Like other enveloped RNA viruses, alphaviruses synthesize and assemble their envelope glycoproteins at the endoplasmic reticulum (ER) membrane. There, protein-conducting channels called translocons provide nascent proteins access to the membrane and allow them to fold into their correct shapes. We previously showed that a hydrophobic segment in the Sindbis virus structural polyprotein forms cotranslational interactions with the translocon that enhance −1 programmed ribosomal frameshifting (−1PRF), a recoding event that regulates polyprotein biogenesis. Recent discoveries concerning translocon remodeling suggest this segment, which corresponds to the second transmembrane domain of the E2 protein, could serve as a signal that recruits the multipass translocon (MPT). Here, we show that knocking out certain components of the MPT increases −1PRF efficiency. These differences in recoding coincide with changes in the membrane topology of the nascent polyprotein and in its downstream proteolytic processing in a manner that ultimately reduces viral fitness. Together, our results indicate that −1PRF and spike protein maturation in alphaviruses is tuned by the dynamic remodeling of the ER translocon. Such coupling could allow polyprotein biogenesis to adapt to different stages of viral replication and to distinct host or vector environments.

Protein secretion routes in fungi are predicted by the length of the hydrophobic helix in the signal sequence

Abstract Secreted proteins are translocated across membranes through multiple routes. In eukaryotes, secreted proteins with N-terminal signal sequences can use either the signal recognition particle and its receptor or the alternative Sec complex to cross the endoplasmic reticulum membrane. Large-scale experiments on the substrates of these pathways are primarily from the model yeast Saccharomyces cerevisiae, but less is known about conservation of translocation pathways. Here, we take a computational approach to analyze secretion signals across the fungal kingdom. Computational predictions by the Phobius model separate secreted proteins in diverse fungal species into distinct populations: cleaved signal peptides with short hydrophobic helices of 8 to 13 amino acids and transmembrane proteins with long hydrophobic helices of 16 to 27 amino acids, similarly to S. cerevisiae. These computational predictions also robustly distinguish translocation routes in S. cerevisiae: Sec-dependent translocation of native proteins is accurately predicted by the presence of a cleaved signal peptide, while conversely signal recognition particle–dependent translocation is predicted by a retained signal-anchor. Analysis of multiple hydrophobicity scales and signal peptide prediction algorithms shows that the Phobius-predicted length of the hydrophobic helix alone is an effective predictor of translocation route. Our results support the hypothesis that the Sec complex is critical for cell wall biogenesis and protein secretion across fungi.

Translation factors that keep the ribosome Pro-active

SUMMARY Ribosomes produce the staggering array of proteins that perform the structural and enzymatic feats of the cell. Therefore, most of the cell’s energy goes toward producing ribosomes and the work performed by them. The work of the ribosome is relatively simple—decode mRNA codons and catalyze the formation of peptide bonds. By marching iteratively along the length of an open reading frame, a complete peptide is produced. Ribosomes catalyze the formation of peptide bonds at a rate of approximately 15 amino acids per second. However, bonds between amino acids do not form with equal efficiency, and ribosomes can become stalled at difficult-to-translate sequences. Proline is unique among the amino acids in that its side chain is covalently bonded to the peptide backbone to form a rigid ring. The rigidity of proline, and especially tracts of proline, makes it a difficult substrate for peptide bond formation, but it is also an essential motif in many protein structures. Elongation factor P (EF-P) is the star player for facilitating translation of polyproline tracts. However, recently identified factors play an important supporting role, and loss of these factors incurs a severe fitness defect in the absence of EF-P. These factors include an EF-P paralog, EfpL, as well as the ABCF ATPase YfmR/Uup and YebC2. The abundance and partial redundancy of factors that prevent ribosome stalling at polyprolines highlights the structural importance of polyproline tracts and the need to facilitate their translation. Here, we review recently identified translation factors that prevent ribosome stalling at polyprolines in bacteria.