Protein Sorting – ER Import, Membrane Insertion & ER Quality Control

These Q&A flashcards cover the full spectrum of lecture 18 topics: ER protein import mechanisms (co- and post-translational), SRP cycle, Sec61 translocon, membrane protein topology, multiple insertion types, tail-anchored pathways, N-linked glycosylation steps, ER chaperones, disulfide bond formation, quality-control cycles, ERAD, and key previous exam questions.

What are the three main events that occur to a protein once it enters the ER lumen?

N-linked glycosylation, disulfide-bond formation/rearrangement, and protein folding assisted by chaperones.

During co-translational translocation, which ribonucleoprotein particle binds the emerging ER-signal sequence?

Signal Recognition Particle (SRP).

Which two GTP-binding partners dock the ribosome–nascent chain complex on the ER membrane?

SRP and the SRP receptor (SR).

What integral membrane channel forms the pore through which polypeptides enter the ER?

The Sec61 translocon complex (Sec61α, β, γ).

Which enzyme removes the ER-signal sequence after entry into the lumen?

Signal peptidase.

Name the ATP-driven chaperone that pulls post-translating polypeptides into the ER lumen.

BiP (an Hsp70 family chaperone).

Which ER membrane complex activates the ATPase activity of BiP during post-translational import?

Sec63 complex.

Are SRP and SRP receptor required for post-translational translocation?

No, they are only required for co-translational translocation.

Define membrane protein topology.

The number and orientation of times a polypeptide spans a membrane.

What sequence halts translocation and anchors Type I single-pass proteins?

Stop-transfer anchor (STA) sequence.

Which side (N- or C-terminus) is luminal for Type I membrane proteins?

N-terminus is luminal; C-terminus faces cytosol.

What determines whether a signal-anchor sequence inserts as Type II or Type III?

The ‘positive-inside’ rule: cluster of positively charged residues remains on the cytosolic side.

Which class of proteins uses a C-terminal hydrophobic segment inserted post-translationally by Get3–Get1/2?

Tail-anchored proteins (e.g., v-SNAREs, t-SNAREs).

What lipid carrier holds the growing N-linked oligosaccharide precursor?

Dolichol phosphate.

List the sugar composition of the full N-linked precursor oligosaccharide.

Glc₃Man₉GlcNAc₂ (3 glucose, 9 mannose, 2 N-acetylglucosamine).

Which antibiotic blocks the first step of N-linked glycosylation?

Tunicamycin.

Which enzyme transfers the oligosaccharide en bloc to Asn residues?

Oligosaccharyl transferase.

State the consensus sequence for N-linked glycosylation.

Asn-X-Ser/Thr where X ≠ Pro.

Where does O-linked glycosylation mainly occur?

Golgi apparatus (also cytoplasm for O-GlcNAc).

Give two functional roles of N-linked glycans on secretory proteins.

Increase solubility and aid in proper folding/quality control; also protect from proteases and mediate cell–cell adhesion.

Name three major ER chaperones involved in glycoprotein folding quality control.

BiP, calnexin, and calreticulin.

Which protein catalyzes disulfide bond formation and rearrangement in the ER?

Protein disulfide isomerase (PDI).

What electron donor re-oxidizes PDI so it can form additional disulfide bonds?

Ero1 (ER oxidoreductin-1).

How are incompletely folded glycoproteins retained in the ER?

By re-addition of one glucose to their N-glycan which allows binding to calnexin/calreticulin.

Which enzyme removes three terminal glucose residues immediately after glycosylation?

Glucosidase I and II (sequentially).

What marks chronically misfolded ER proteins for ER-associated degradation (ERAD)?

Mannose trimming (to Man5/6) followed by recognition by OS-9, dislocation, ubiquitination, and proteasomal degradation.

Which chaperone recognizes free sulfhydryl groups and prevents exit of incompletely folded proteins?

ERp57 (also called GRp58).

Differentiate N-linked and O-linked glycosylation by linkage type.

N-linked has β-N-glycosidic bond to Asn amide nitrogen; O-linked has α-O-glycosidic bond to hydroxyl of Ser/Thr/Hyp/Hyl.

Which quality-control step acts as the protein-folding ‘sensor’ in ER?

A single mannose removal by mannosidase after Glc removal, producing Man8 glycan.

Name the exam-asked statement that correctly describes proteins translated on rough ER.

They are proteins targeted to lysosomes, plasma membrane, or secretion (cell exterior).

What type of intracellular trafficking from ER to Golgi is NOT transmembrane transport?

Vesicular transport (budding and fusion).

SRP stalls translation by blocking which stage?

Elongation of the polypeptide chain.

Which structural feature of SRP binds the hydrophobic signal sequence?

Signal-sequence binding domain (SSBD) with hydrophobic groove.

How many proteins and RNA molecules compose mammalian SRP?

Six proteins and one ~300-nt RNA (7SL RNA).

What is the energy source for SRP–SR docking and release?

GTP binding and hydrolysis on both SRP and SR.

Which amino-acid characteristic defines transmembrane α-helices?

20–25 consecutive hydrophobic residues forming an α-helix.

Explain the ‘positive-inside rule’.

Positively charged residues adjacent to a signal-anchor stay on the cytosolic side, determining orientation.

Give an example of a Type I membrane protein.

Low-density lipoprotein (LDL) receptor.

Give an example of a Type II membrane protein.

Transferrin receptor.

Give an example of a Type III membrane protein.

Cytochrome P450.

Multipass Type IV proteins include which well-known receptor family?

G-protein-coupled receptors (GPCRs).

Which proteins are GPI-anchored after cleavage of a C-terminal signal peptide?

Examples include plasminogen activator receptor and Fasciclin II.

Why is N-linked glycosylation important for many viral envelope glycoproteins?

It confers folding assistance, immune evasion, and protease resistance (e.g., influenza HA).

What happens to the Sec61 translocon when a stop-transfer sequence exits laterally into the bilayer?

The channel closes and the polypeptide is laterally released to anchor the protein.

Which pathway inserts proteins into chloroplasts and mitochondria after complete synthesis in cytosol?

Post-translational transmembrane transport via TOM/TIM (mitochondria) or TOC/TIC (chloroplasts) channels.