Project description:The guided entry of tail-anchored proteins (GET) pathway assists in the proper delivery of tail-anchored (TA) proteins to the ER. Here we uncover how the yeast GET pathway components Get4/5 mediate capture of TA proteins by Sgt2, which interacts with TA sequences and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of TA proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting of proteins containing internal TM domains. Exposure of nascent, internal TM domains at the tunnel exit induces high-affinity ribosome-binding of SRP, which in turn prevents ribosome-binding of Get4/5. In this way, the position of TM domains within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.
Project description:Proteomic analysis was performed on ventricular tissues isolated from embryonic day 14.5 (E14.5) cardiomyocyte-specific Asna1 conditional knockout mice and littermate controls. Heart lysates were digested with Trypsin/Lys-C and analyzed by LC-MS/MS using an EASY-nanoLC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Label-free quantification (LFQ) was used to identify differentially expressed proteins associated with tail-anchored protein targeting, vesicular trafficking, and ER-Golgi transport pathways.
Project description:Enhanced ascorbate peroxidase 2 (APEX2) can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein, ideally within a confined compartment. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. Here we used rapamycin-dependent targeting of APEX2 to tail-anchored proteins of the endoplasmic reticulum and of the inner nuclear membrane (INM). In combination with stable isotope labeling with amino acids in cell culture (SILAC), our novel approach (rapamycin- and APEX-dependent identification of proteins by SILAC, or RAPIDS) allowed the identification of proteins that are in close proximity to the prototypic INM-protein emerin and the vesicle-trafficking protein VAPB. In addition to well-known interaction partners, several new potential binding partners were identified. In RAPIDS, the comparison of plus/minus-rapamycin conditions allows a clear distinction between specific and non-specific hits.
Project description:Ribosome hibernation is a commonly used strategy that protects ribosomes under unfavorable conditions and regulates developmental processes. Multiple ribosome-hibernation factors have been identified in all domains of life, but due to their structural diversity and the lack of a common mechanism by which they inactivate ribosomes, it is currently unknown how many different hibernation factors exist. Here, we demonstrate that the YqjD/ElaB/YgaM protein family constitute an abundant class of ribosome-hibernating proteins and represent the first membrane-bound hibernating factors identified in bacteria. Our data demonstrate that they interact with 50S ribosomal subunit and show a concentration-dependent inhibition of protein synthesis. By combining in vivo cross-linking with mass spectrometry, we show that they bind via their N-termini to proteins that surround the ribosomal tunnel exit and even penetrate into the ribosomal tunnel. Thus, YqjD/ElaB/YgaM inhibit translation by blocking the ribosomal tunnel and thus mimic the activity of antimicrobial peptides and macrolide antibiotics.
Project description:The loss of the tail is among the notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the “anthropomorphous apes”, with a hypothesized role in contributing to human bipedalism. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here, we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element – inserted into an intron of the TBXT gene – pairs with a neighboring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of mouse Tbxt, mimicking the expression pattern of its hominoid ortholog TBXT. We found that mice expressing both Tbxt isoforms can exhibit a complete absence of the tail or a shortened tail, depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud, supporting the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. We further noted that mice expressing the exon-skipped Tbxt isoform may develop neural tube defects, a condition that affects ~1/1,000 neonates in human. Thus tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.