Chapter 13: Moving Proteins into Membranes and Organelles

Compartmentalization of Cells

Introduction to Protein Targeting

Cells are highly organized structures with distinct compartments, each performing specialized functions. Proteins must be accurately delivered to their correct locations within the cell to maintain cellular function. This process involves several mechanisms for moving proteins between compartments, including gated transport, transmembrane transport, and vesicular transport.

• Gated Transport: Selective gates (e.g., nuclear pore complexes) allow the regulated movement of macromolecules between the cytosol and nucleus.

• Transmembrane Transport: Protein translocators facilitate the movement of proteins across membranes into organelles such as the ER, mitochondria, and peroxisomes.

• Vesicular Transport: Membrane-enclosed vesicles shuttle proteins between organelles of the endomembrane system.

Protein Synthesis and Initial Localization

Sites of Protein Synthesis

Most cellular proteins are synthesized by ribosomes in the cytosol. A small subset is synthesized within mitochondria or plastids (in plants).

• Cytosolic Ribosomes: Synthesize the majority of proteins, which may remain in the cytosol or be targeted to other compartments.

• Mitochondrial/Plastid Ribosomes: Synthesize a few organelle-specific proteins.

Cell Membrane Permeability

Barriers to Protein Movement

The lipid bilayer of cellular membranes is selectively permeable. Most proteins cannot passively diffuse across membranes and require specialized transport mechanisms.

• Small Molecules: Can diffuse freely or via channels.

• Proteins: Require active transport or translocation mechanisms.

Evolution of Organelles

Distinct Organelle Families

Cellular organelles are classified into four evolutionary families based on their structure and function:

• Nucleus and Cytosol: Continuous via nuclear pore complexes (NPC).

• Endomembrane System: Includes ER, Golgi, lysosomes, and vesicles.

• Mitochondria and Peroxisomes: Specialized for energy metabolism and detoxification.

• Plastids: Found only in plants, involved in photosynthesis and storage.

Protein Sorting and Targeting

Sorting Signals

Proteins contain intrinsic signals within their amino acid sequences that dictate their final destination. These sorting signals are essential for proper cellular organization.

• Signal Sequences: Short stretches (15-60 amino acids), often at the N- or C-terminus, recognized by cellular machinery.

• Signal Patches: Multiple internal sequences forming a 3D structure.

• No Sorting Signal: Protein remains in the cytosol.

Examples of Signal Sequences

• ER Targeting: N-terminal sequence with 5-10 hydrophobic amino acids.

• Nuclear Localization Signal (NLS): Short, positively charged sequence (rich in lysine or arginine).

• Mitochondrial Targeting: Amphiphilic helix with alternating positive and hydrophobic residues.

• Peroxisomal Targeting: Three characteristic amino acids at the C-terminus (e.g., Ser-Lys-Leu).

Physical properties (hydrophobicity, charge placement) are often more important than the exact sequence.

Manipulation of Sorting Signals

• Sorting signals are necessary and sufficient for targeting.

• Artificial addition or removal of signals can redirect protein localization.

• Example: Nucleoplasmin (nuclear protein) with NLS localizes to nucleus; removal of NLS or addition to another protein alters localization.

Mechanisms of Protein Transport

Gated Transport

Occurs through selective gates such as nuclear pore complexes, allowing regulated exchange between nucleus and cytosol.

Transmembrane Transport

Proteins are translocated across membranes into organelles like ER, mitochondria, and peroxisomes via protein translocators.

Vesicular Transport

Proteins are packaged into vesicles for transport between organelles of the endomembrane system (ER, Golgi, lysosomes, etc.).

Endoplasmic Reticulum (ER) and Protein Translocation

Structure of the ER

The ER is a network of membranes with rough (ribosome-studded) and smooth regions. It is the entry point for proteins destined for secretion, membranes, or other organelles.

Types of Proteins Entering the ER

• Transmembrane Proteins: Partially translocated and embedded in the membrane.

• Water-Soluble Proteins: Fully translocated into the ER lumen, destined for secretion or residence in the lumen.

Most ER import is co-translational (occurs during protein synthesis), but post-translational import also occurs, especially in yeast.

Signal Recognition and Translocation

• Signal Sequence: 16-30 amino acids, mostly non-polar.

• SRP (Signal Recognition Particle): Binds the signal sequence and docks the ribosome to the ER membrane.

• Translocon: Channel with 10 membrane-spanning helices, opens for protein entry during translation.

• BiP: Chaperone that powers post-translational import using ATP.

Transmembrane Protein Topology

• Hydropathy Profiles: Used to predict transmembrane domains based on hydrophobicity.

• Topological Classes: Even number of TM domains = termini on same side; odd number = opposite sides.

• GPI-Anchored Proteins: Glycosylphosphatidylinositol anchors allow for freer movement in the membrane.

• C-Tail Anchored Proteins: Inserted via Get3 ATPase, independent of SRP.

Protein Processing in the ER

Major Modifications

• Glycosylation: Covalent addition of sugars, forming glycoproteins. N-linked glycosylation occurs on asparagine residues; O-linked occurs on serine/threonine.

• Disulfide Bond Formation: Catalyzed by protein disulfide isomerase (PDI), stabilizes protein structure.

• Proteolytic Cleavage: Removal of signal sequences and activation of proteins.

• Folding and Assembly: Chaperones (e.g., calnexin, calreticulin) assist in proper folding and multi-subunit assembly.

Glycosylation Process

• Dolichol: Lipid carrier for oligosaccharide assembly, flipped into ER lumen for transfer to proteins.

• Further Modification: Oligosaccharides are processed in the Golgi apparatus.

• Function: Glycans serve as tags for folding, quality control, and cell signaling.

Quality Control and Disease

ER Quality Control

• Lectins: Bind incompletely folded glycoproteins, retaining them in the ER for refolding or degradation.

• Unfolded Protein Response (UPR): Cellular response to ER stress, can lead to apoptosis if misfolded proteins accumulate.

• Example: Cystic fibrosis results from misfolded membrane protein discarded by ER quality control.

Transport into Other Organelles

Mitochondria

• Precursor Proteins: Synthesized in cytosol, remain unfolded until import.

• Signal Sequence: Amphiphilic helix directs to mitochondria.

• Translocators: TOM (outer membrane), TIM (inner membrane) complexes mediate import, powered by chaperones and ATP.

Chloroplasts

• Similar import mechanism to mitochondria, involving TIC and TOC complexes.

• Proteins have N-terminal signal sequences, removed after import.

Peroxisomes

• Import Signal: C-terminal tripeptide (Ser-Lys-Leu).

• Import Mechanism: Post-translational, proteins can be folded during import, mediated by peroxins (Pex proteins) and ATP.

• Disease Example: Zellweger syndrome results from mutations in Pex genes, leading to defective peroxisomal protein import.

Nuclear Transport

Nuclear Pore Complex (NPC)

• Structure: ~2000 NPCs per cell, composed of nucleoporins.

• Transport: Passive diffusion for small molecules (<5 kDa), active transport for larger proteins and RNAs (up to 40 kDa).

• FG-Repeats: Phenylalanine-glycine repeats create a gel-like matrix for selective transport.

Nuclear Import and Export

• Importins: Recognize NLS, shuttle cargo through NPC.

• Exportins: Recognize nuclear export signals (NES), mediate export of large molecules.

• Energy Requirement: Transport is fueled by GTP hydrolysis via Ran GTPase.

• Regulation: Import/export rates can be modulated by masking signals or post-translational modifications (e.g., phosphorylation).

Summary Table: Protein Targeting Signals and Destinations

Destination	Signal Sequence	Key Features
Endoplasmic Reticulum	N-terminal, 5-10 hydrophobic aa	SRP recognition, co-translational import
Nucleus	NLS (short, basic aa)	Importins, NPC transport
Mitochondria	Amphiphilic helix	TOM/TIM complexes, post-translational import
Peroxisome	C-terminal SKL	Peroxins, folded protein import
Chloroplast	N-terminal, amphiphilic	TIC/TOC complexes, post-translational import

Key Equations and Concepts

• Hydropathy Index: Used to predict transmembrane domains. $H = \sum_{i=1}^{n} h_i$ where $h_i$ is the hydrophobicity value of amino acid $i$.

• GTP Hydrolysis in Nuclear Transport: $\text{Ran-GTP} \rightarrow \text{Ran-GDP} + \text{P}_i$

Conclusion

Accurate protein targeting and transport are essential for cellular function and health. Understanding the mechanisms and signals involved in protein movement between compartments provides insight into cell biology and the basis of many diseases.