BIOC 406 Exam 3

What are the two major types of protein transport?

Post-translational transport (translation finishes in the cytosol, then protein is directed elsewhere) and co-translational transport (ribosome docks onto the rough ER during translation).

Where does translation of most proteins begin?

In the cytosol, regardless of whether the protein will ultimately stay there or be transported elsewhere.

What determines which transport pathway a protein takes?

Specific amino acid sequences ('zipcodes') in the protein's sequence, which are recognized by dedicated machinery.

What is the 'zipcode' for nuclear import?

Nuclear localization sequence (NLS) — a cluster of basic (positively charged) amino acids.

What is the 'zipcode' for nuclear export?

Nuclear export sequence (NES) — a hydrophobic sequence rich in leucine residues.

How do small vs. large proteins pass through the nuclear pore complex (NPC)?

Proteins <40 kDa can diffuse passively through the FG diffusion barrier; proteins ≥40 kDa require importins, exportins, and the Ran GTPase.

What is the 'zipcode' for mitochondrial matrix import?

An N-terminal amphipathic helix (the mitochondrial targeting sequence, MTS) that is recognized by the TOM complex.

What happens to the mitochondrial targeting sequence after import?

It is cleaved off by a protease inside the mitochondrial matrix.

What is the 'zipcode' for peroxisomal import?

A C-terminal SKL (Ser-Lys-Leu) sequence, known as the peroxisome targeting sequence (PTS).

Which proteins recognize and deliver cargo to peroxisomes?

PEX5 recognizes the SKL cargo and delivers it to PEX13 at the peroxisomal membrane, which has a YG diffusion barrier.

What machinery mediates mitochondrial import?

The TOM complex (Translocase of the Outer Membrane) recognizes the MTS; a motor then translocates the protein across the inner membrane.

What triggers a ribosome to switch from cytosolic translation to co-translational (ER) transport?

The emerging polypeptide contains an ER signal sequence, which is recognized by the Signal Recognition Particle (SRP).

What are the four sequential steps of co-translational transport?

1. Initiation — translation begins in cytosol. 2. Recruitment — SRP binds ER signal sequence, translation pauses, ribosome docks at ER via SRP receptor. 3. Translocation — ribosome binds Sec61/preprotein translocase, translation resumes into ER. 4. Release — ribosomal subunits recycled to cytosol.

What happens to translation when SRP binds the ER signal sequence?

Translation pauses (elongation arrest) until the SRP-ribosome complex docks onto the SRP receptor on the ER membrane.

In the correct order, place these co-translational events: (I) SRP binds SRP receptor, (II) SRP binds ER signal sequence, (III) protein synthesis pauses, (IV) protein synthesis resumes, (V) Sec61 binds ER signal sequence.

II → III → I → V → IV. (SRP binds signal → pauses translation → docks at ER receptor → Sec61 engages signal → translation resumes with translocation.)

What is the secretory pathway and what final destinations does it serve?

The route proteins take from the rough ER → Golgi → vesicles → final destinations, which include the plasma membrane, ER, Golgi, lysosomes, and the peroxisomal membrane.

What are the key features of an ER signal sequence (signal peptide)?

A largely hydrophobic stretch of 6–15 amino acids, often preceded by a positively charged residue near the N-terminus; may contain a cleavage site for signal peptidase.

What is the function of the positively charged residue flanking the hydrophobic core of the ER signal sequence?

It helps orient the signal peptide correctly within the Preprotein Translocase (Sec61) of the ER membrane — consistent with the positive-inside rule.

What is the KDEL sequence and what is its function?

Lys-Asp-Glu-Leu — a C-terminal tetrapeptide that signals retention in (or retrieval back to) the ER lumen.

What cleaves the ER signal sequence after translocation, and where does cleavage occur?

Signal peptidase, an enzyme located in the ER membrane on the lumenal side, cleaves the signal sequence to release the mature protein.

Do all ER signal sequences get cleaved?

No. Only those with an appropriate cleavage site are cleaved by signal peptidase; some signal sequences remain embedded in the membrane (e.g., as transmembrane anchors in Type II proteins).

What is the SRP in eukaryotes?

A large ribonucleoprotein (RNP) made of RNA and multiple protein subunits (including SRP54, SRP19, SRP9/14, SRP68/72). It is sometimes called the 'third component of the ribosome.'

What are the three functions of SRP?

1. Recognize the ER signal sequence emerging from the ribosome exit tunnel. 2. Pause translation (elongation arrest). 3. Direct the ribosome and nascent polypeptide to the Sec61 translocon on the ER membrane.

How does SRP physically pause translation?

SRP spans from the peptide exit site to the elongation-factor-binding site of the ribosome in a kinked conformation, sterically blocking elongation.

What is the SRP receptor and where is it located?

A membrane protein on the cytosolic face of the rough ER membrane; SRP delivers the paused ribosome to it, allowing translocation to begin.

Why is SRP required for the secretory pathway?

It is the essential factor that recognizes ER signal sequences and co-translationally redirects ribosomes to the rough ER; without it, secretory/membrane proteins would be fully translated in the cytosol.

What is the preprotein translocase of the ER, and what subunits compose it?

The heterotrimeric Sec61 complex, composed of α, β, and γ subunits.

How does the Sec61 complex span the ER membrane?

It contains 12 transmembrane helices that form a protein-conducting channel (pore) from the cytosolic face to the ER lumen.

What structural features of the Sec61 complex mediate protein transport?

A movable plug domain (lumenal side, gates the pore) and a lateral gate (facing the lipid bilayer, allows insertion of TMDs into the membrane).

What conformational changes occur in Sec61 when the ER signal sequence arrives?

The signal sequence interacts with the lateral gate, causing: (1) displacement of the plug domain (opening the pore) and (2) opening of the lateral gate toward the lipid bilayer.

What is the TRAP complex and how does it relate to Sec61?

The Translocase Associated Protein (TRAP) complex associates with Sec61 and is thought to promote the open (active) state of the Sec61 channel.

What is OST and what does it do in the context of the Sec61 complex?

Oligosaccharyl Transferase (OST) associates with the Sec61 complex and glycosylates (adds N-linked sugars to) proteins co-translationally as they emerge into the ER lumen.

What happens after a soluble protein is fully translocated through Sec61?

Signal peptidase cleaves the ER signal sequence, releasing the mature protein into the ER lumen; OST adds N-linked glycans; the protein then folds.

What is a Type I membrane protein?

A single-pass membrane protein with its N-terminus in the ER lumen (signal peptide cleaved) and C-terminus in the cytosol. Insertion involves a stop-transfer/TMD sequence that exits through Sec61's lateral gate.

What is a Type II membrane protein?

A single-pass membrane protein with its N-terminus in the cytosol and C-terminus in the ER lumen. The TMD acts like a signal peptide to open the Sec61 channel; no cleavage step occurs.

What is the 'positive-inside rule'?

The positively charged residues flanking a transmembrane domain are always found on the cytosolic side of the membrane, regardless of N- or C-terminus orientation.

Is the positive-inside rule dependent on whether the charged residues are at the N- or C-terminus?

No — it is entirely independent of which terminus carries the charge. It applies to the charges flanking each individual TMD.

What is a stop-transfer sequence?

A hydrophobic TMD that, when emerging from the ribosome during translocation, exits through Sec61's lateral gate and anchors the protein in the ER membrane, halting further translocation.

How does a multipass (polytopic) membrane protein get inserted?

Multiple TMDs are threaded through the Sec61 channel sequentially; each exits through the lateral gate into the lipid bilayer in turn, driven by the hydrophobicity of each TMD.

What tool can predict how many times a protein spans the membrane?

A hydropathy plot — peaks above the hydrophobicity threshold indicate putative transmembrane helices (TMDs).

What information does a hydropathy plot provide?

It plots the average hydropathy index along the amino acid sequence; hydrophobic peaks (above ~+1.6) suggest transmembrane helices, indicating how many times the protein crosses the membrane.

What is glycosylation?

The covalent attachment of sugar molecules to a protein — a major post-translational (and co-translational) modification that affects folding, stability, targeting, and function.

What is N-linked glycosylation and where does it occur?

Attachment of a 14-residue oligosaccharide to the nitrogen of an Asn side chain; occurs co-translationally in the ER by OST, at the sequon Asn-X-Ser/Thr (X ≠ Pro).

What is the glycosylation sequon (signal) for N-linked glycosylation?

Asn – X – Ser/Thr, where X can be any amino acid except Pro.

How are N-linked glycans added?

By 'block transfer' — OST transfers a preassembled 14-residue oligosaccharide (containing glucose, mannose, N-acetylglucosamine) en bloc to the Asn side chain.

What is O-linked glycosylation and where does it occur?

Attachment of sugars to the oxygen of Ser or Thr residues; occurs in the Golgi by the serial addition/removal of monosaccharides by glycosyltransferases and glycosidases on completed polypeptides.

List four major functions of glycosylation.

Protein folding, protein stability, protein targeting (e.g., M6P for lysosomes), cell–cell adhesion, immune evasion/recognition, and cell–cell signaling.

What are ABO blood groups an example of?

Glycosylation — the A and B antigens are glycoproteins whose sugar modifications differ between blood types.

Who won the 2022 Nobel Prize in Chemistry related to glycosylation research?

Carolyn Bertozzi, for developing click chemistry and bioorthogonal chemistry to study cell-surface glycosylation and its roles in cancer, inflammation, and bacterial infection.

In the secretory pathway, what modifications occur in the ER?

N-linked glycosylation, disulfide bond formation (between Cys pairs), proteolytic cleavages, protein folding, protein quality control, and packaging into COPII vesicles for transport to the Golgi.