Protein Processing, Folding, and Secretion in Bacteria L17 EXAM 2

Protein Processing, Secretion, and Targeting

Introduction to Protein Processing

Proteins often require processing before they become functional within the cell. This processing can involve folding, incorporation of cofactors, or targeting to specific cellular locations. Proper protein processing is essential for cellular activities and survival.

• Protein Processing: May include assistance in folding or the incorporation of cofactors or other nonprotein groups.

• Targeting: Proteins must be directed to particular cellular locations such as membranes, the periplasm, or even outside the cell.

• Secreted Proteins: Some proteins (e.g., toxins, extracellular enzymes) are secreted from the cell to function in the environment or affect other cells.

• Processing and Targeting Mechanisms: Often require internal "signal" sequences within the protein and/or accessory proteins (chaperones) that assist with folding and transport.

Assisted Protein Folding and Chaperones

Role and Function of Chaperones

While many proteins can fold spontaneously into their secondary, tertiary, and quaternary structures, others require assistance from chaperones. Chaperones are specialized proteins that catalyze macromolecular folding events and are highly conserved across all domains of life.

• Functions of Chaperones:

  • Helping proteins with initial folding

  • Refolding partially denatured proteins

  • Untangling RNAs

  • Incorporating cofactors into enzymes

• Conservation: Chaperone sequences are highly conserved among all organisms.

Key Chaperones in E. coli

In Escherichia coli, several key chaperones facilitate protein folding, especially under stress conditions.

• DnaK and DnaJ: ATP-dependent enzymes that bind newly formed polypeptides and slow folding to increase the likelihood of correct folding.

• GroEL and GroES: If the DnaKJ complex cannot fold the protein properly, it may transfer it to GroEL and GroES. GroEL is barrel-shaped and uses energy from ATP hydrolysis to fold the protein with the assistance of GroES.

• Essentiality: Of the several thousand proteins in an E. coli cell, many require GroEL-GroES to fold properly; a subset of these proteins is essential for cell survival.

Chaperones and Stress Responses

Chaperones also play a critical role in refolding proteins that become partially denatured due to environmental stress, such as temperature changes.

• Heat Shock Proteins: Chaperones are a type of heat shock protein involved in the heat shock response, where cells attempt to refold partially denatured proteins rather than degrade them.

• Regulation: Heat shock protein synthesis is upregulated under high temperatures, while cold shock protein synthesis is upregulated at low temperatures.

• RNA Chaperones: Cold affects RNAs more than proteins, inducing production of RNA chaperones as well as some protein chaperones.

• Cofactor Assembly: Some chaperones help assemble cofactor-containing enzymes, such as those involved in redox and electron transport chain reactions.

Protein Secretion: The Sec and Tat Systems

Translocases and Protein Transport

Proteins called translocases are responsible for transporting specific proteins through bacterial and archaeal membranes. Two major systems, Sec and Tat, facilitate this process.

• Sec Translocase System: Exports unfolded proteins and inserts integral membrane proteins into the cytoplasmic membrane.

• Tat Translocase System: Transports previously folded proteins through the cytoplasmic membrane.

• Signal Sequence: Most proteins that must be transported through membranes contain a signal sequence (15-20 residues long; positively charged residues followed by hydrophobic residues, then polar residues). This sequence is found at the N-terminus of membrane/secreted proteins and signals the secretory system to translocate the protein. It also prevents the protein from completely folding, which could interfere with secretion.

Mechanism of the Sec System

• Recognition: In the Sec system, unfolded proteins are recognized by SecA protein (binds proteins for export to the periplasm) and the signal recognition particle (SRP), which binds proteins that are inserted into the cytoplasmic membrane.

• SRP Composition: In bacteria, SRPs consist of a single protein and a small noncoding RNA.

• Delivery: Both SecA and SRP deliver proteins to the membrane secretion complex.

• Signal Sequence Removal: After transport/delivery, the signal sequence is removed by a protease, allowing the protein to complete folding.

Mechanism of the Tat System

• Requirement: Some proteins must be transported outside the cell after they have been folded, often because they contain cofactors incorporated during folding.

• Tat System: The Tat (twin arginine translocase) system transports these folded proteins. Proteins have a signal sequence with a pair of arginine residues recognized by TatBC proteins, which carry the protein to TatA, the membrane transporter.

• Signal Sequence Removal: Following transport, the signal sequence is removed by a protease.

Protein Secretion: Gram-Negative Secretion Systems

Types I-VI Secretion Systems

Gram-negative bacteria possess multiple specialized secretion systems (Types I-VI) to transport proteins or effectors across their complex cell envelope, which includes both an inner and outer membrane.

• Function: These systems insert proteins or effectors into the outer membrane, secrete them outside the cell, or inject them into a recipient cell.

• Gram-Positive Bacteria: Use similar systems, but only need to transport proteins across the cytoplasmic membrane.

• Facilitated Activities: Secretion systems are involved in symbiosis, biofilm formation, enzyme secretion, DNA transfer, antibiotic release, and protein delivery.

• Structure: Each system is composed of a large complex of proteins that specifically recognize its substrate and form a translocase channel spanning one or more membranes.

Mechanisms of Secretion Systems

• One-Step Systems: Types I, III, IV, and VI use a channel through both membranes for direct transport.

• Two-Step Systems: Types II and V require initial transport through the inner membrane (often via Sec or Tat), followed by a second group of transporters to move the protein through the outer membrane.

• Injection Mechanism: Some systems function like a syringe to inject molecules into host cells.

Summary Table: Comparison of Major Bacterial Secretion Systems

System Type	Transport Mechanism	Substrate State	Membranes Traversed	Example Function
Sec	Translocase (SecA/SRP)	Unfolded	Inner membrane	General protein export
Tat	Twin arginine translocase	Folded (with cofactors)	Inner membrane	Export of cofactor-containing proteins
Type I, III, IV, VI	One-step channel	Varied	Inner & outer membranes	Effector injection, toxin secretion
Type II, V	Two-step (Sec/Tat + outer membrane transporter)	Varied	Inner & outer membranes	Enzyme secretion, biofilm formation

Example: Type III secretion system in pathogenic Salmonella functions like a molecular syringe to inject virulence factors directly into host cells.

Additional info: The notes infer the essential role of chaperones and secretion systems in bacterial physiology, especially in adaptation to environmental stress and pathogenesis.