#protein targeting

Cell Organelles

The N-terminal signal sequence of secretory proteins is recognized by:

Targeting of secretory proteins to endoplasmic reticulum uses signal hypothesis where N-terminal fifteen to thirty amino acid hydrophobic sequence emerges from ribosome exit tunnel exposing binding site for signal recognition particle, a three hundred kDa ribonucleoprotein comprising seven SL RNA and six protein subunits including SRP54 that contains methionine-rich pocket accommodating hydrophobic signals via induced fit. Binding transiently arrests elongation, complex diffuses to ER membrane where heterodimeric SRP receptor composed of SR alpha and SR beta, both GTPases, docks particle. GTP hydrolysis by SRP54 and SR alpha drives transfer of signal sequence to Sec61 translocon channel, opening laterally for membrane integration. Ribosome then resumes translation translocating nascent chain into lumen. SNARE proteins mediate post-targeting vesicle fusion using coiled-coil zippering, clathrin forms lattice coats during endocytosis, ribophorin anchors ribosome but does not recognize signal. Genetic ablation in bacteria Ffh or yeast SRP54 leads to mislocalization of secreted proteins aggregating in cytosol, confirming essential sorting role preventing toxic accumulation.

Ref: Walter Nature 1981 SRP discovery; SRP54 binds signal peptide, GTP-dependent delivery to Sec61.

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Protein Sorting and Trafficking

What signal is required for targeting proteins to the thylakoid lumen?

Final intra-chloroplast sorting thylakoid lumen after stroma entry requires crossing thylakoid membrane barrier maintaining proton gradient ATP synthesis. Lumenal precursors plastocyanin copper OE16 OE23 photosystem II oxygen evolving complex Rieske iron-sulfur PetC transport stroma lumen. Precursors carry bipartite targeting sequences N-terminal chloroplast transit peptide TOC-TIC entry second thylakoid transfer domain 30-70 residues positively charged N-region hydrophobic core C-region cleavage thylakoid processing peptidase. Two distinct pathways recognize transfer domain: Sec pathway transports unfolded chains SecYE channel driven SecA ATPase proteins plastocyanin apoprotein folding after transport Tat twin-arginine translocation transports fully folded proteins already assembling cofactors disulfide bonds stroma cofactor insertion before transport essential unfolded impossible. Diagnostic signature Tat substrates twin-arginine motif SRRXFLK invariant consecutive arginines RR N-region signal plus hydrophobic Phe-Leu-Lys essential recognition cpTatC Hcf106 receptor complex oligomeric pore varied size driven exclusively proton motive force Delta pH across thylakoid not ATP. SKL peroxisomal PTS1 matrix NPXY endocytic internalization PTB adaptors DXE ER export COPII unrelated thylakoid lumen sorting. Mutation arginine pair lysine abolishes import demonstrating motif necessity and energy coupling mechanism distinct.

Ref: Cline & Theg, Mol Membr Biol 2007: Twin-arginine RR motif for Tat pathway to thylakoid lumen.

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Protein Sorting and Trafficking

Which pathway is involved in the targeting of thylakoid proteins?

Thylakoid membrane harbors photosynthetic machinery requiring insertion light-harvesting polytopic proteins after chloroplast envelope import stroma. Four conserved pathways inherited cyanobacterial ancestor: Sec pathway unfolded proteins SecYEG translocase SecA ATPase, SRP pathway specialized highly hydrophobic light-harvesting chlorophyll a/b binding proteins LHCPs pigments. Stromal chloroplast SRP cpSRP54 GTPase homologous bacterial SRP54 NG M domains binding signal sequence GTP, unique cpSRP43 chaperone absent bacteria three chromodomains four ankyrin repeats specifically binding L18 motif 18 amino acids between transmembrane helices two three LHCP preventing aggregation maintaining soluble transit competent essential pigments otherwise aggregate uncontrollably. Cargo-cpSRP complex targets thylakoid membrane via cpFtsY GTPase homologous FtsY SRalpha then hands substrate Alb3 insertase Oxa1 YidC family integrating bilayer lateral gate GTP hydrolysis dependent. OXA pathway mitochondria inserts inner proteins matrix side TIM23 handles presequence translocation mitochondrial inner membrane MIA40 mediates oxidative disulfide mitochondrial IMS none relevant thylakoid. Loss cpSRP43 Arabidopsis chaos pale green phenotype severe loss LHCPs demonstrating indispensable role thylakoid assembly functional chloroplast development and photosynthesis efficiency and light harvesting.

Ref: Schuenemann, Annu Rev Plant Biol 2007: cpSRP pathway targets proteins to thylakoid membrane.

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Protein Sorting and Trafficking

Proteins destined for the intermembrane space of mitochondria follow which pathway?

Intermembrane space houses many small proteins twin cysteine motifs serving redox sensing copper delivery assembly such as small Tim family Tim9 Tim10 Tim13 Cox17 copper chaperone cytochrome oxidase Erv1 sulfhydryl oxidase Mia40 receptor. Most contain CX3C or CX9C helical hairpin stabilized two disulfide bonds. Import after TOM entry IMS relies dedicated MIA pathway mitochondrial IMS import and assembly comprising oxidoreductase Mia40 CHCHD4 mammals hydrophobic binding cleft redox-active CPC disulfide sulfhydryl oxidase Erv1 ALR FAD cofactor. Mechanism oxidative folding trap: Mia40 recognizes reduced unfolded substrate hydrophobic cleft forming transient intermolecular mixed disulfide via substrate cysteine attack Mia40 CPC; subsequent attack second substrate cysteine introduces intramolecular disulfide substrate concomitant release oxidized folded substrate reduced Mia40. Erv1 reoxidizes Mia40 disulfide transferring electrons FAD cytochrome c respiratory chain completing catalytic cycle generating disulfide. Trapping mechanism does not require membrane potential or ATP unlike TIM23 motor resembles ER PDI-Ero1 but mitochondria specific. TOM-MIA pathway defines IMS proteome via disulfide relay ensuring retention and functional folding quality control and biogenesis.

Ref: Herrmann & Riemer, Curr Opin Cell Biol 2012: MIA40 pathway for IMS protein oxidative folding.

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Protein Sorting and Trafficking

Which protein assists in inserting tail-anchored proteins into the ER membrane?

Tail-anchored proteins pose topological challenge because single transmembrane domain near extreme C-terminus, often within last thirty residues, emerges from ribosome only after termination codon, precluding co-translational SRP recognition. Cellular solution is Guided Entry of Tail-anchored proteins, GET pathway. Newly released tail-anchored client is captured in cytosol by pre-targeting complex Sgt2-Get4-Get5 that hands hydrophobic tail to dimeric ATPase Get3 in yeast, TRC40 in mammals, forming closed dimer that shields tail via methionine-rich groove similar to SRP54. ATP binding closes dimer protecting tail from aqueous environment preventing aggregation. Complex docks to ER membrane receptors Get1-Get2, mammalian orthologs WRB and CAML, multi-pass membrane proteins that insert tail into bilayer using energy from ATP hydrolysis triggering Get3 opening and release. This pathway handles many SNAREs such as synaptobrevin, syntaxin-5, and apoptosis regulators Bcl-2. SRP recognizes N-terminal signals during translation, Ran controls nuclear import, BiP works lumenally as ratchet, so Get3/TRC40 defines distinct post-translational pathway dedicated to C-terminal membrane insertion and organelle targeting specificity for essential cellular functions. Additional coordination with cellular stress pathways ensures fidelity, prevents aggregation, and links trafficking to growth control and proteostasis maintenance across diverse cell types and developmental stages.

Ref: Hegde & Keenan, Nature Rev Mol Cell Biol 12: 2011, GET Pathway for Tail-Anchored Insertion.

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Protein Sorting and Trafficking

Which domain of SRP binds to signal sequences on nascent proteins?

Specificity of signal recognition particle for hydrophobic targeting signals resides within C-terminal M domain of 54 kilodalton subunit SRP54. Structural analyses of bacterial homolog Ffh and mammalian SRP54 show M domain folds into deep groove lined almost exclusively with methionine side chains whose flexible thioether and long aliphatic chain create plastic hydrophobic bristle adaptable to varied signal sequence compositions and lengths. Basic residues surrounding groove interact with phosphate backbone of 7SL RNA and ribosomal proteins L23 and L29 near peptide exit tunnel. Adjacent NG GTPase domains dimerize with SRP receptor SRα via GTP-dependent interaction but do not contact signal directly. Upon signal accommodating as alpha-helix inside groove, conformational change extends to linker connecting M and NG domains, signaling Alu domain to pause translation. ATPase or BiP domains absent from SRP. This methionine-rich architecture explains how single particle binds hundreds of diverse ER targeting signals with high affinity yet promiscuous selectivity, ensuring efficient capture of secretory proteins early during synthesis preventing cytosolic mislocalization, aggregation and degradation by proteasome quality control and maintaining secretory flux.

Ref: Keenan et al., Annu Rev Biochem 70: 2001, SRP54 M Domain Binds Signal Sequences.

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Protein Sorting and Trafficking

The function of the SRP (Signal Recognition Particle) is to:

Signal recognition particle integrates targeting and translation control to ensure secretory proteins avoid premature cytosolic folding and aggregation. Particle consists of 7SL RNA scaffold folding into Alu and S domains and six proteins. Most critical subunit SRP54 contains N domain four-helix bundle, central GTPase domain and C-terminal M domain rich in methionines that forms deep groove accommodating diverse hydrophobic signal sequences through flexible sulfur-containing side chains providing plastic hydrophobic surface adaptable to many sequences. Binding occurs when signal emerges from ribosome, inducing structural rearrangement transmitted to Alu domain formed by SRP9/14 heterodimer and RNA hairpin that interacts with elongation factor binding site near GTPase center of ribosome, slowing elongation by competing with incoming aminoacyl-tRNA. This pause extends time window for diffusion to ER membrane where heterodimeric SRP receptor SRα-SRβ, both GTPases, captures complex via GTP-dependent dimerization of NG domains. GTP hydrolysis drives hand-off of ribosome-nascent chain to Sec61 and recycles SRP. COPII transport, BiP ATPase activation and vesicle docking are unrelated to primary SRP activity of recognition and pausing, ensuring fidelity and targeting efficiency.

Ref: Egea et al., Curr Opin Struct Biol 15: 2005, SRP Function in Signal Recognition and Pause.

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Protein Sorting and Trafficking

Which of the following recognizes the signal sequence of nascent proteins targeting the ER?

Early sorting of nascent secretory and membrane proteins is performed by signal recognition particle, conserved ribonucleoprotein containing 7SL RNA scaffold and six protein subunits. When hydrophobic signal sequence of about eight to twelve non-polar residues emerges from ribosomal exit tunnel, methionine-rich M domain of SRP54 forms flexible hydrophobic groove whose abundance of sulfur-containing methionine side chains allows plastic accommodation of diverse signal sequences via induced fit. Binding is communicated through 7SL RNA to Alu domain comprised of SRP9 and SRP14 heterodimer that docks at elongation factor binding site, temporarily pausing translation to prevent premature folding and aggregation in cytosol. SRP-ribosome-nascent chain complex then diffuses to ER where GTP-dependent interaction with heterodimeric SRP receptor made of SRα and SRβ GTPases delivers complex to Sec61 channel for hand-off. Ran-GTP controls nuclear import via importins, KDEL receptor retrieves escaped ER chaperones via COPI, Rab GTPases govern vesicle tethering and fusion specificity, none directly scan ribosome exit tunnel for hydrophobic nascent signals at this early checkpoint of protein sorting to ER lumen.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 13: SRP Recognizes Signal Sequences.

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Protein Sorting and Trafficking

What is the role of signal sequences in protein sorting?

Protein sorting fidelity in eukaryotic cells depends on specific topogenic signals encoded within polypeptide sequences acting as molecular zip codes recognized by targeting machinery. N-terminal signal peptide typical for secretory pathway contains approximately 15 to 30 amino acids organized into positively charged N-region with basic residues, central hydrophobic H-region of 7 to 15 leucine, valine, isoleucine residues forming alpha-helix, and C-region with polar residues and Ala-X-Ala motif for signal peptidase cleavage. Upon emergence from ribosome exit tunnel, hydrophobic core bound by 54 kDa subunit of signal recognition particle SRP that pauses translation and delivers ribosome-nascent chain complex to SRP receptor heterodimer at rough ER via GTP hydrolysis cycle, transferring chain to Sec61 translocon heterotrimer. Signal inserts into lateral gate opening channel, translocation proceeds cotranslationally into ER lumen or integration into membrane. Other signals include nuclear localization signal with lysine rich clusters, mitochondrial amphipathic helix, and peroxisomal SKL tripeptide, each ensuring accurate compartmentalization.

Ref: Blobel & Dobberstein, J Cell Biol 1975, Signal Hypothesis. Alberts 7th ed., Chapter 12, Targeting Signals.

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