Biomedical subjects

Yuanlong Shao

Publications and source records attributed to Yuanlong Shao.

6 recordsLinked to original sources

PubMed2005

The cotranslational contacts between ribosome-bound nascent polypeptides and the subunits of the hetero-oligomeric chaperonin TRiC probed by photocross-linking.

The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (delta and either beta or epsilon), actin and luciferase chains photocross-link to at least six TRiC subunits (alpha, beta, delta, epsilon, xi, and theta) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits zeta and theta was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold.

Actins↗

PubMed2004

Cotranslational integration and initial sorting at the endoplasmic reticulum translocon of proteins destined for the inner nuclear membrane.

The current diffusion-retention model for protein trafficking to the inner nuclear membrane (INM) proposes that INM proteins diffuse laterally from the membrane of the endoplasmic reticulum into the INM and are then retained in the INM by binding to nuclear proteins or DNA. Because some data indicate that the sorting of baculovirus envelope proteins to the INM is protein-mediated, we have examined the early stages of INM protein integration and sorting by using photocrosslinking. Both viral and host INM-directed proteins were integrated cotranslationally through the endoplasmic reticulum translocon, and their nonrandom photocrosslinking to two translocon proteins, Sec61alpha and translocating chain-associated membrane protein (TRAM), revealed that the first transmembrane sequence (TMS) of each viral and host INM-directed protein occupied a very similar location within the translocon. Because few TMSs of non-INM-directed membrane proteins photocrosslink to TRAM, it seems that the INM-directed TMSs occupy different sites within the translocon than do non-INM-directed TMSs. The distinct proximities of translocon components to INM-directed TMSs strongly suggest that such TMSs are recognized and initially sorted within the translocon. Taken together, these data indicate that membrane protein sorting to the INM is an active process involving specific nonnuclear proteins.

Amino Acid Sequence↗

PubMed2004

Bcl-2 homodimerization involves two distinct binding surfaces, a topographic arrangement that provides an effective mechanism for Bcl-2 to capture activated Bax.

The homo- and heterodimerization of Bcl-2 family proteins is important for transduction and integration of apoptotic signals and control of the permeability of mitochondria and endoplasmic reticulum membranes. Here we mapped the interface of the Bcl-2 homodimer in a cell-free system using site-specific photocross-linking. Bcl-2 homodimer-specific photoadducts were detected from 11 of 17 sites studied. When modeled into the structure of Bcl-2 core, the interface is composed of two distinct surfaces: an acceptor surface that includes the hydrophobic groove made by helices 2 and 8 and the loop connecting helices 4 and 5 and a donor surface that is made by helices 1-4 and the loop connecting helices 2 and 3. The two binding surfaces are on separate faces of the three-dimensional structure, explaining the formation of Bcl-2 homodimers, homo-oligomers, and Bcl-2/Bax hetero-oligomers. We show that in vitro the Bcl-2 dimer can still interact with activated Bax as a larger oligomer. However, formation of a Bax/Bcl-2 heterodimer is favored, since this interaction inhibits Bcl-2 homodimerization. Our data support a simple model mechanism by which Bcl-2 interacts with activated Bax during apoptosis in an effective manner to neutralize the proapoptotic activity of Bax.

Apoptosis↗

PubMed2003

Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex.

In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61alpha and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST. No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.

Amino Acid Sequence↗

PubMed2003

Signal recognition particle binds to ribosome-bound signal sequences with fluorescence-detected subnanomolar affinity that does not diminish as the nascent chain lengthens.

The binding of signal recognition particle (SRP) to ribosome-bound signal sequences has been characterized directly and quantitatively using fluorescence spectroscopy. A fluorescent probe was incorporated cotranslationally into the signal sequence of a ribosome.nascent chain complex (RNC), and upon titration with SRP, a large and saturable increase in fluorescence intensity was observed. Spectral analyses of SRP and RNC association as a function of concentration allowed us to measure, at equilibrium, K(d) values of 0.05-0.38 nm for SRP.RNC complexes with different signal sequences. Competitive binding experiments with nonfluorescent RNC species revealed that the nascent chain probe did not alter SRP affinity and that SRP has significant affinity for both nontranslating ribosomes (K(d) = 71 nm) and RNCs that lack an exposed signal sequence (K(d) = 8 nm). SRP can therefore distinguish between translating and nontranslating ribosomes. The very high signal sequence-dependent SRP.RNC affinity did not decrease as the nascent chain lengthened. Thus, the inhibition of SRP-dependent targeting of RNCs to the endoplasmic reticulum membrane observed with long nascent chains does not result from reduced SRP binding to the signal sequence, as widely thought, but rather from a subsequent step, presumably nascent chain interference of SRP.RNC association with the SRP receptor and/or translocon.

Animals↗

PubMed2003

Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins.

During cotranslational protein integration into the ER membrane, each transmembrane (TM) segment moves laterally through the translocon to reach the lipid bilayer. Photocrosslinking studies reveal that a particular surface of each nascent chain TM alpha helix and signal-anchor sequence always faces Sec61alpha in the translocon. This nonrandom and TM sequence-dependent positioning reveals that each TM segment makes specific contacts with Sec61alpha and is retained at a fixed location within the translocon, observations that are best explained by the binding of each TM sequence to a translocon protein(s). Since TM sequence hydrophobicity does not correlate with its rate of release from the translocon, nascent chain movement through the translocon appears to be mediated primarily by protein-protein interactions rather than hydrophobic nascent chain-phospholipid interactions.

Bacterial Proteins↗