lecture 6 Protein Transport and Translocation Mechanisms

SRP and Ribosome Interaction

• The Signal Recognition Particle (SRP) attaches to its receptor, bringing the SRP-ribosome complex to a protein translocator in the endoplasmic reticulum (ER) membrane.
• Once bound, the part of SRP near the ribosomal tunnel repositions, allowing the translocator to take its place.
• This process enables the translocator to transfer the growing protein across the membrane, creating two distinct populations of ribosomes: free ribosomes and membrane-bound ribosomes.

Polyribosome Formation

• Multiple ribosomes can simultaneously engage with a single mRNA, forming what is called a polyribosome.
• Once a polyribosome is attached to the ER membrane, the individual ribosomes can return to the cytosol upon completing translation, while the mRNA remains anchored at the ER.

Translocator Structure

• The translocator forms a water-filled channel that allows the polypeptide chain to pass through.
• The core structure, called the Sec61 complex, consists of three conserved subunits from bacteria to eukaryotes and contains 10 helices surrounding a central channel.
• A short helix plugs this channel when the translocator is idle, preventing ion leakage.
• The Sec61 translocator is selectively activated only for proteins with a signal sequence.

Signal Sequence Recognition

• Observations from cryo-electron microscopy reveal how the signal sequence of a protein engages with the Sec61 translocator.
• The N-terminus of the signal sequence, when inserted into the lateral gate of Sec61, triggers the opening of the central channel by releasing the plug and allowing the polypeptide to be threaded through the channel.
• This translocation occurs concurrently with protein translation.

Post-translational Translocation

• Some proteins, completely synthesized in the cytosol, undergo post-translational translocation into the ER as unfolded polypeptides, due to the narrow channel of Sec61.
• Chaperone proteins, such as the hsp70 family, prevent these polypeptides from folding prematurely.
• The signal peptide of a precursor directly engages the Sec61 translocator to open the channel, facilitated by accessory proteins that use energy for translocation.

Insertion of Transmembrane Proteins

• Transmembrane proteins have hydrophobic segments recognized like signal sequences during their insertion into the ER membrane.
• The orientation of the transmembrane segment during insertion depends on the positioning of charged amino acids and the N-terminus's length and stability.
• A cleaved N-terminal signal sequence directs initial translocation, while hydrophobic segments later insert into the lateral gate, halting translocation.

Multipass Transmembrane Proteins

• Multipass transmembrane proteins span the membrane multiple times, with each hydrophobic segment alternating in orientation based on the preceding segments.
• The first segment’s insertion establishes a topology that governs the orientation of subsequent transmembrane segments.

Protein Folding and Assembly in the ER Lumen

• Proteins synthesized in the rough ER often contain an ER retention signal at their C-terminus to remain in the ER.
• Chaperones, like BiP, assist in correctly folding proteins, while Protein disulfide isomerase (PDI) aids in the formation of disulfide bonds, and can reverse incorrect bonding.

Glycosylation in the ER

• A key biosynthetic function of the ER is protein glycosylation, specifically N-linked glycosylation, where oligosaccharides are covalently attached to an asparagine residue on the protein.
• This process involves the transfer of a preformed oligosaccharide precursor from dolichol to proteins, mediated by oligosaccharyl transferases.

Monitoring Protein Folding via Oligosaccharides

• Newly synthesized proteins undergo oligosaccharide trimming; glucosidases remove glucose residues initially attached.
• The singly glucosylated oligosaccharide binds to calnexin and calreticulin, which assist in folding. Incorrectly folded proteins may have glucose re-added to aid in refolding.

ER Export and Cytosolic Degradation of Improperly Folded Proteins

• The trim of mannose on the oligosaccharide serves as a timer for proteins in the ER. Special lectins recognize improperly folded proteins for retrotranslocation.
• Translocators with E3 ubiquitin ligases attach polyubiquitin tags for degradation via the proteasome in the cytosol.

Overview of Nuclear Transport

• The nuclear envelope consists of two membranes with nuclear pore complexes (NPCs) that function as selective gates for molecules moving between cytosol and nucleus.
• Different transport mechanisms include protein translocation, gated transport, and vesicular transport, each facilitating the movement of specific molecules or organelles.

Nuclear Pore Complex Structure

• NPCs are composed of 30 different nucleoporins forming a central pore and are capable of transporting large macromolecules.
• The sieve-like interior of the NPC ensures selective permeability, allowing small molecules to pass freely while restricting larger ones.

Nuclear Localization Signals

• Proteins with nuclear localization signals (NLSs) rely on karyopherins for active transport into the nucleus. NLSs are rich in basic amino acids and can be found anywhere in the protein sequence.
• Fully-folded proteins, unlike denatured polypeptides, are actively imported into the nucleus through NPCs.

Ran GTPase and Directionality of Nuclear Transport

• Ran GTPase, existing in GTP and GDP-bound states, regulates the destination of nuclear transport. High Ran-GTP in the nucleus promotes cargo release from import receptors.
• In contrast, Ran-GDP in the cytosol allows import receptors to bind to new cargo.

Nuclear Export Dynamics

• Nuclear export involves the selective transport of molecules via nuclear export signals, dependent on binding to specific export receptors.
• The conditions for cargo binding differ in import and export, thus affecting how and where proteins interact with the transport machinery.

Regulation of Nuclear Transport

• Cells modulate the rates of nuclear import and export through mechanisms like phosphorylation of import signals, controlling access to the transport machinery, and triggering changes in binding state for transcription regulators.

Example of Regulated Nuclear Import During T Cell Activation

• In resting T cells, NF-AT is phosphorylated and cytosolic, but upon T cell activation, increased intracellular calcium dephosphorylates NF-AT, exposing NLSs and permitting nuclear entry.

SREBP and Cholesterol Biosynthesis Regulation

• SREBP serves as a transcription factor for cholesterol biosynthesis, maintaining inactivity in the ER until needed. In low cholesterol conditions, SREBP can be activated and transported to the Golgi for processing into its active form.