Protein Sorting and Trafficking

Protein Sorting and Trafficking

What is the primary function of Pex11 in peroxisomes?

Peroxisomes are single membrane organelles handling beta oxidation of very long chain fatty acids, alpha oxidation, plasmalogen synthesis and H2O2 detoxification via catalase. Their abundance adapts to metabolic demand through fission of preexisting organelles rather than solely de novo budding from ER. Pex11 family proteins, including Pex11 alpha beta gamma isoforms in mammals, are conserved peroxisomal membrane proteins enriched in regions of high curvature. Upon induction by oleate, fibrates via PPAR alpha, or cold, PEX11 genes transcribed, proteins oligomerize through amphipathic helices a

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 13: Peroxisome Biogenesis and Pex11 Division.

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Protein Sorting and Trafficking

What happens when the transferrin receptor is mutated and cannot bind Fe³⁺ at acidic pH?

Iron uptake in proliferating cells depends on transferrin receptor mediated endocytosis tightly coupled to endosomal acidification. At extracellular pH 7.4 holo transferrin carrying two ferric ions binds transferrin receptor 1 homodimer with nanomolar affinity, clusters via AP2 clathrin adaptor and internalizes into early endosomes. Vacuolar ATPase acidifies lumen to about pH 5.5, protonation of transferrin histidines and nearby receptor residues induces conformational opening of transferrin lobes, reducing Fe3+ affinity by orders of magnitude and releasing ferric iron while apo transferrin re

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 13: Endocytosis and Transferrin Iron Transport.

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Protein Sorting and Trafficking

Which of the following best describes the role of LC3 in autophagy?

LC3 proteins are mammalian homologs of Saccharomyces Atg8, central ubiquitin like modifiers governing autophagosome membrane dynamics. Newly synthesized LC3 is cleaved by ATG4 family proteases exposing C terminal glycine to generate cytosolic LC3-I. Upon autophagy induction triggered by starvation or rapamycin, ATG7 E1 activates LC3-I, transfers to ATG3 E2, and ATG12 ATG5 ATG16L1 complex acting as E3 ligase conjugates LC3 to phosphatidylethanolamine on nascent isolation membrane, forming LC3-II. Lipidated LC3-II integrates into both inner and outer leaflets, promoting membrane hemifusion, elon

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 12: Autophagy and LC3 Lipidation Mechanism.

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Protein Sorting and Trafficking

Which inhibitor blocks autophagosome-lysosome fusion?

Autophagic flux progresses through initiation, elongation, closure, fusion and degradation. Efficient cargo clearance requires autophagosome lysosome fusion mediated by small GTPase RAB7, HOPS complex and SNARE proteins STX17 SNAP29 VAMP8 and YKT6, plus tethering factors like PLEKHM1. Bafilomycin A1, a macrolide isolated from Streptomyces griseus, is a high affinity inhibitor of vacuolar type H+ ATPase V0 domain c subunit, blocking proton pumping into lysosome lumen. Collapse of acidic pH inactivates cathepsins and prevents LysoTracker accumulation. Recent work shows additional target ER calci

Ref: NCBI Bookshelf, Molecular Biology of the Cell, Section: Autophagosome-Lysosome Fusion and V-ATPase Inhibitors.

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Protein Sorting and Trafficking

Which process is activated during nutrient deprivation?

During prolonged starvation cells must recycle internal components to maintain energy and biosynthetic precursors. In fed state mTORC1 kinase anchored at lysosome via Rag GTPases phosphorylates ULK1 and ATG13, keeping initiation complex inactive. When amino acids fall, Rag switches off, mTORC1 dissociates, inhibitory phosphorylation is lost. Concurrently low ATP raises AMP, activating AMPK which phosphorylates ULK1 at distinct activating sites Ser317 Ser777 and inhibits mTORC1 via TSC2 and Raptor. Free ULK1 complex phosphorylates Beclin-1 and ATG14L, activating VPS34 class III PI3K to produce

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 12: Intracellular Vesicular Traffic and Autophagy Regulation.

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Protein Sorting and Trafficking

What is the primary function of amphisomes?

Interplay macroautophagy and endocytic system produces hybrid intermediate called amphisome fusion product before lysosomal degradation. Autophagosome double-membrane vesicle size up to 1 micron bearing lipidated LC3-II cargo sequestered including mitochondria protein aggregates bacteria forms upon closure isolation membrane phagophore. Separately endocytic pathway internalizes surface receptors extracellular material into early endosomes marked Rab5 PI3P then matures Rab7 positive late endosomes multivesicular bodies MVBs intraluminal vesicles tetraspanins CD63 LAMP1 LBPA. Amphisome formation

Ref: Nakamura & Yoshimori, Autophagy 2017: Amphisome formation from autophagosome-endosome fusion.

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Protein Sorting and Trafficking

Which protein mediates retrograde transport of ER proteins from the Golgi?

Maintaining ER proteome against continuous leakage via bulk flow into Golgi relies active retrieval preventing depletion resident chaperones folding enzymes. ER membrane proteins expose C-terminal dilysine motif KKXX or KXKXX with two lysines at -3 -4 positions relative extreme C-terminus recognized directly WD40 beta-propeller domains COPI subunits alpha-COP beta prime-COP binding dilysine pH independent. Soluble ER luminal residents like BiP HSPA5 GRP94 PDI calreticulin bear C-terminal KDEL; after escape bind KDEL receptor family Erd2 homologs KDELR1-3 seven transmembrane proteins concentrat

Ref: Bethune et al., Cold Spring Harb Perspect Biol 2006: COPI retrieval of ER proteins from Golgi.

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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 trans

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

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

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Protein Sorting and Trafficking

What is the main function of the TIC complex in chloroplasts?

Chloroplast envelope encloses two translocon systems tandem importing majority organelle proteome 3000 proteins despite plastid genome about 100. Precursors synthesized cytosol N-terminal transit peptide enriched hydroxylated serine threonine positively charged amphipathic not hydrophobic distinct mitochondrial presequence. First receptor TOC outer envelope: Toc159 large 159 kDa GTPase selectivity filter photosynthetic versus housekeeping via acidic A-domain, Toc34 small 34 kDa regulatory, Toc75 Omp85 beta-barrel superfamily 14-strand channel 3 nm pore translocating polypeptide. After outer me

Ref: Soll & Schleiff, Nat Rev Mol Cell Biol 2004: TIC imports proteins across chloroplast inner envelope.

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Protein Sorting and Trafficking

Which protein is required for peroxisomal membrane biogenesis?

De novo biogenesis and growth peroxisomal membrane depends targeted insertion peroxisomal membrane proteins PMPs specialized machinery distinct matrix pathway. PMPs including docking Pex14 Pex13 fission factors Pex11 isoforms transporters ABCD1-3 synthesized free cytosolic ribosomes containing hydrophobic transmembrane domains aggregation prone requiring chaperoning. Cytosolic receptor chaperone Pex19 farnesylated C-terminal CAAX box binds PMPs hydrophobic groove preventing aggregation maintaining solubility delivering peroxisomal membrane docking platform. Targeting essential factors Pex3 42

Ref: Distel et al., Annu Rev Biochem: Pex3 and Pex16 essential for peroxisomal membrane biogenesis.

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Protein Sorting and Trafficking

What happens when Pex5 is ubiquitinated by Pex2, Pex10, and Pex12?

Import receptor recycling distinguishes peroxisomes mitochondria requiring extraction shuttling receptor after cargo delivery. After releasing matrix cargo lumen transient pore Pex5 remains embedded peroxisomal membrane peripherally N-terminus facing cytosol partial retrotranslocation. Return cytosol new cycles membrane extraction requires ubiquitination generating handle AAA motor. Specialized peroxisomal E3 ligase complex RING proteins Pex2 Pex10 Pex12 heterotrimeric zinc coordinated embedded membrane associates cytosolic E2 Pex4 anchored membrane Pex22. Pex5 undergoes thioester monoubiquiti

Ref: Platta et al., Cell Cycle 2007: Pex5 monoubiquitination by Pex2/10/12 recycles receptor via Pex1/6.

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