BIOS 222 Chapter 11

Chapter 11: Protein Trafficking into the Endoplasmic Reticulum

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Learning Objectives

• Understand the components of the endomembrane system.

• Comprehend the evolution of the endomembrane system.

• Recognize the similarities between some organelle lumens and the extracellular environment.

• Understand protein movement through the endomembrane system.

• Differentiate between rough and smooth endoplasmic reticulum (ER).

• Understand the shape of the ER.

• Recognize the continuity between the ER and the outer nuclear membrane, including ribosomes located on the outer nuclear membrane.

• Overview the translation of proteins into the rough ER.

Introduction to the Endomembrane System

Anatomy, Evolution, Environment, and Movement

The Endomembrane System

• Overall Structure:

  • The endomembrane system begins at the Endoplasmic Reticulum (ER) and progresses through the Golgi Apparatus.

• Possible End Destinations:

  • Endosomes

  • Lysosomes

  • Peroxisomes

  • Cell Membrane

• Trafficking:

  • Trafficking vesicles facilitate the movement between organelles and membranes.

Endomembrane Evolution

• Foundation:

  • Likely originated from plasma membrane invagination (though this is still debated).

• Supportive Evidence:

  • The double membrane structure of the nuclear envelope.

  • The outer nuclear membrane being continuous with the ER.

Environmental Similarities

Chemical Characteristics of Organelle Lumens

• Some organelle lumens exhibit chemical similarities to the extracellular matrix.

  • Identified as a “non-cytosolic environment.”

  • Example organelles include the endoplasmic reticulum and Golgi apparatus.

• Contrast with Cytosol:

  • These lumens are markedly different from the cytosolic environment.

• Protein Trafficking:

  • Most proteins trafficked via the endomembrane system ultimately reside in the extracellular solution/cell membrane or within endomembrane compartments such as endosomes and lysosomes.

  • Note: The lumen refers to the interior cavity of organelles or tubular structures.

Protein Movement through the Endomembrane System

• Outward Movement:

  • RNA is transported from the nucleus to the rough ER.

  • Proteins move from the ER to the Golgi apparatus.

  • Proteins in vesicles are transported from the Golgi to various organelles and membranes.

• Resident Proteins:

  • Certain proteins (resident proteins) are meant to remain in the ER or Golgi and will not continue to progress through the system.

• Regulatory Signals:

  • Protein trafficking through the endomembrane system is tightly controlled and initiated by signal sequences within proteins.

• Translation in the Rough ER:

  • Proteins destined for the endomembrane system are translated on the ER, entering the ER where they undergo chemical modifications and, barring the resident ER proteins, are eventually packaged into vesicles and transported to the Golgi for further modification and sorting.

The Golgi “Airport” Analogy

• The movement and processing of proteins can be likened to an airport:

  • Protein Passengers:

  • Proteins arrive and progress through the rough ER.

  • Security Check:

  • The transition from ER to Golgi is like a security check—only properly folded proteins can proceed.

  • Signal Sequence Tickets:

  • These help proteins reach the correct 'gates' at the Golgi for sorting.

  • Airport Staff Analogy:

  • Resident ER and Golgi proteins are likened to airport staff.

  • Shipping Mechanism:

  • Vesicles are represented as “airplanes” that transport proteins to their final destinations.

Entering the Rough ER

Rough vs Smooth ER, ER Signal Sequence, Signal Recognition Particle, Soluble vs Transmembrane Proteins

Rough and Smooth ER

• ER Description:

  • The ER is the largest organelle within cells, composed of an extensive membrane network.

• Rough ER Characteristics:

  • Continuous with the outer nuclear membrane.

  • Composed of flat cisternae.

  • Main site for protein synthesis, indicated by ribosome coverage—hence the name “rough.”

• Smooth ER Characteristics:

  • Made up of tubular structures.

  • Functions as a site for lipid synthesis; its smooth surface indicates an absence of ribosomes.

• Interconnected Structure:

  • The cisternae and tubules are interconnected, encapsulating a singular internal space.

ER Shape

• Shape Maintenance:

  • The distinct shapes of the ER are maintained by specific proteins.

  • Example:

  • CLIMP-63 extends between the sides of cisternae to retain a flat configuration.

  • Other proteins embedded in the outer phospholipid layer curve the edges of the cisternae.

Continuity of ER and Nuclear Membrane

• Structural Continuity:

  • The ER is continuous with the outer nuclear membrane, which, in turn, is continuous with the inner nuclear membrane.

  • Nuclear Pore Complexes (NPCs):

  • NPCs regulate protein movement between the inner and outer nuclear membranes, thus preventing unintended crossings.

Ribosomes on the Outer Nuclear Membrane

• Ribosome Function:

  • The outer nuclear membrane features ribosomes due to its continuity with the ER.

  • These ribosomes are responsible for translating soluble proteins into the perinuclear space (area between the inner and outer nuclear membranes) and for translating membrane proteins that are embedded in the outer nuclear membrane.

  • Translated proteins migrate into the rough ER lumen (continuous with the perinuclear space) and into the rough ER membrane itself.

Overview of Translation of Proteins into the Rough ER

• Signal Recognition Particle (SRP):

  • A SRP binds to the emerging N-terminal of the protein being translated in the cytosol.

  • The ribosome-SRP complex is subsequently recruited to an SRP receptor located on the rough ER membrane.

  • The ribosome is then passed to a translocation channel known as a translocon.

  • Soluble polypeptides are directly translated into the rough ER lumen, whereas membrane polypeptides are similarly translated but include one or more stop signals that facilitate protein embedding in the membrane.

Learning Objectives (Revisited)

• Understand how proteins enter the rough ER during translation.

• Explain how nascent proteins affiliate with the rough ER, including the functions of ER signal sequences, SRP, SRP receptor, and translocon.

• Discuss how soluble proteins are processed upon entry into the ER lumen.

• Describe the processing of membrane-embedded proteins in the rough ER, including differences for single and multi-pass membrane proteins.

• Outline the contribution and role of chemical modifications, particularly focusing on disulfide bonds and glycosylation.

• Define glycoproteins and their structural and functional characteristics.

• Analyze the glycosylation process of proteins entering the ER.

Entry of Proteins into Endomembrane System

• Mechanism of Entry:

  • Proteins enter the endomembrane system as they are translated in the rough ER by ribosomes located on the cytosolic surface of the rough ER and the outer nuclear membrane.

  • This process is referred to as co-translational translocation.

Choosing the ER Pathway

• Translation Initiation:

  • mRNA translation typically begins on a ribosome in the cytoplasm.

• Determining Pathways:

  • The N-terminus of the peptide determines the pathway—either the cytosolic pathway or the endomembrane pathway.

  • If the N-terminus contains an ER signal sequence, translation pauses;

  • ER signal sequences are approximately 15-30 amino acids long and are notably hydrophobic.

Signal Recognition Particle (SRP)

• Composition and Function:

  • The ER signal sequence activates recruitment of a signal recognition complex (SRP).

  • SRP consists of six proteins that are bound to RNA:

  • One end contains a hydrophobic pocket to bind the hydrophobic ER signal sequence.

  • The opposite end blocks the ribosome entrance, halting translation temporarily to prevent tRNAs from entering.

  • The SRP/polypeptide/ribosome complex binds to an SRP receptor embedded in the rough ER membrane.

Recruitment of SRP to the Rough ER

• Complex Binding:

  • The ribosome-SRP complex interacts with the SRP receptor in the rough ER membrane.

  • Both SRP and the SRP receptor possess bound GTP; upon binding, this GTP is hydrolyzed.

  • Importance of GTP Hydrolysis:

  • GTP hydrolysis facilitates the transfer of the ribosome to the rough ER translocon channel.

  • This hydrolysis acts as a checkpoint ensuring that the ribosome disengages from the SRP only at the rough ER membrane, where the SRP receptors reside.

Soluble Proteins

• Process of Translation:

  • When the ribosome attaches to the translocon, the ER signal sequence on the polypeptide undergoing translation interacts with the translocon, prompting the opening of the central channel.

  • The ribosomal exit tunnel aligns with this channel, allowing translation to resume.

  • As synthesis occurs, the polypeptide is translocated into the ER luminal side, a process termed co-translational translocation.

  • Signal Peptidase Function:

  • This enzyme cleaves the ER signal sequence, facilitating the release of the polypeptide into the ER lumen.

Single-Pass Transmembrane Proteins

• Binding Mechanism:

  • Similar to the soluble proteins, the ER signal sequence in single-pass transmembrane proteins initially binds to the translocon (which will later be cleaved).

  • The polypeptide chain then threads through the membrane into the rough ER lumen.

• Additional Signal Requirement:

  • Membrane polypeptides feature an additional stop signal, inhibiting the entire polypeptide from traversing the rough ER membrane.

  • The stop signal comprises hydrophobic residues interacting with hydrophobic residues within the channel, preventing further passage.

  • Final Configuration:

  • N-terminal end resides in the rough ER lumen, C-terminal end remains in the cytosol, and the transmembrane domain spans the translocon channel.

  • Upon translation completion, the transmembrane domain is released to the lateral region of the rough ER membrane.

Multi-Pass Transmembrane Proteins

• Features of Multi-Pass Proteins:

  • Hydrophobic start and stop signal sequences function in pairs to weave the protein into the membrane.

  • In some cases, the initial signal sequence is an internal signal which is not cleaved; it thus remains embedded in the membrane.

Covalent Modifications in the Rough ER

Disulfide Bond Formation and Glycosylation

Covalent Modifications in the ER

• Covalent modifications initiate immediately upon protein entry into the ER.

• Types of Modifications:

  • Disulfide bonds and glycosylation occur solely on non-cytosolic proteins.

  • These modifications protect proteins against harsh non-cytosolic conditions, such as proteolytic activity, and extreme fluctuations in pH, temperature, mechanical stress, and oxidizing environments.

• Functional Roles:

  • Disulfide bonds afford structural stability and can prevent protein unfolding.

  • Glycosylation serves as a shield against proteolytic degradation, augments thermostability, and increases solubility.

Disulfide Bond Formation

• Definition:

  • Disulfide bonds are covalent links formed between sulfur atoms from two proximal cysteine residues.

  • These results can occur within a single polypeptide chain (at tertiary structure).

  • Alternatively, they can form between distinct polypeptide chains (at quaternary structure).

• Catalysis:

  • This modification is facilitated by the enzyme protein disulfide isomerase (PDI) within the rough ER lumen.

Functions of Disulfide Bonds

• These bonds protect proteins from oxidation by stabilizing their structures in environments susceptible to oxidative conditions.

• Oxidizing Environment:

  • The presence of oxygen can disrupt protein structures through electron removal, altering protein functionality.

  • The cytosol acts as a reducing environment, contrasting with the extracellular space. - Formation of covalent bonds between cysteine sulfur atoms secures electrons, safeguarding protein integrity against oxidative alterations.

Glycosylation

• Definition:

  • Glycosylation involves the covalent addition of an oligosaccharide, resulting in the formation of glycoproteins.

• Prevalence:

  • The majority of proteins entering the ER (both soluble and membrane-bound) undergo glycosylation.

• Location of Activity:

  • This modification occurs on the luminal side of the ER.

• Protective Nature:

  • Functions at both the protein and cellular level as a barrier and recognition feature in multicellular organisms.

Attaching the First Oligosaccharide

• First Oligosaccharide:

  • A 14-sugar oligosaccharide is the initial identifier for glycosylation.

  • It comprises three glucose, nine mannose, and two N-acetylglucosamine units.

  • Attachment:

  • This oligosaccharide initially binds to a dolichol lipid on the ER membrane.

  • Transfer Mechanism:

  • The process of transfer to proteins commences through the enzyme oligosaccharyl transferase, linking to the glycosylation signal Asn-X-Ser/Thr.

  • Attachment occurs at the nitrogen of Asn—this process is termed N-linked glycosylation, and it represents an early event after the protein emerges into the ER lumen.

Preparing to Journey through the Endomembrane System

The Four Checkpoints, Asymmetrical Membranes, and Proteins

Learning Objectives (Revisited)

• Comprehend that ER exit is controlled to guarantee protein quality.

• Recognize the five checkpoints utilized to ensure correct protein folding and modification.

• Understand the implications of having unfolded or misfolded proteins accumulate, which can lead to cell death or diseases.

• Acknowledge that proteins correctly folded in the ER exit via ER exit sites (ERESs).

• Identify the role of chaperones in retaining improperly assembled proteins in the ER.

• Discuss functionality and nuances of the five checkpoints that inhibit the release of non-qualifying proteins from the ER.

Exit from the ER and Selectivity

• The exit from the ER is highly selective:

  • Proteins failing to fold correctly or that do not assemble properly (e.g., dimers or multimers) are actively retained in the ER by chaperone proteins.

  • Accumulation of improperly folded proteins incurs risks of cell death and diseases such as cancer, diabetes, and neurodegenerative conditions in humans.

The 1st Checkpoint: Leaving the ER

• Mechanism of Exit:

  • Both soluble proteins in the rough ER lumen and membrane proteins in the rough ER membrane exit through designated ER exit sites (ERESs).

  • Only properly folded and modified proteins are allowed to depart via ERESs.

  • The first checkpoint assesses that all proteins are adequately folded and, if necessary, properly covalently modified.

• Packaging in Vesicles:

  • Soluble and membrane protein packages occur within ERESs for subsequent transport to the Golgi.

  • Soluble proteins maintain their soluble state in the Golgi, while rough ER membrane proteins integrate into the Golgi membrane.

Quality Control: Chaperone Proteins

• Role of Chaperones:

  • Operate at the first checkpoint within the ERES.

  • Key chaperones include BiP and Calnexin, which bind to misfolded proteins.

  • BiP is a soluble chaperone in the ER lumen.

  • Calnexin is an integral membrane protein located in the ER membrane, with its N-terminal in the lumen.

  • Both proteins specifically cling to exposed hydrophobic amino acids in unfolded proteins, delaying their exit until properly folded.

  • Outcome:

  • Only properly folded proteins can proceed to leave the ERES.

The 2nd Checkpoint: Getting Sent Back

• Forward Movement:

  • Transition from the rough ER to the Golgi is predominantly anterograde.

  • Quality Control Mechanism:

  • Occasionally, improperly folded or modified proteins escape the rough ER control and enter Golgi-bound vesicles inadvertently.

  • Such proteins are identifiable within the Golgi and are reverted to the rough ER via retrograde transport vesicles.

  • This return mechanism constitutes the second quality control checkpoint.

  • ER Retention Signal:

  • The return process for resident ER proteins necessitates an ER retention signal, like KDEL, which interacts with specific membrane receptors within the Golgi.

The 3rd Checkpoint: Unfolded Protein Response (UPR)

• Trigger Mechanism:

  • If a high proportion of misfolded proteins arises, the unfolded protein response (UPR) is triggered.

  • UPR is indirectly activated by chaperone BiP, which typically binds to Ire1 and misfolded proteins.

  • Elevated misfolded protein levels result in diminished BiP availability for Ire1 binding, leading to formation of Ire1 dimers.

  • Signaling Cascade:

  • Ire1 initiates a signaling cascade that boosts production of additional chaperones, quality control proteins, and promotes ER membrane expansion.

The 4th Checkpoint: Dislocation

• Continued Accumulation:

  • If misfolded proteins persist despite the UPR's attempts to rectify the situation, dislocation is enacted.

  • This process involves exporting misfolded proteins from the rough ER through a membrane channel.

  • Mechanisms underlying dislocation are not deeply understood.

  • Tagging for Degradation:

  • Misfolded proteins are ubiquitinated for subsequent degradation via the proteasome system.

  • Consequences of Failure:

  • A breakdown within this dislocation checkpoint may lead to apoptosis (programmed cell death) and contribute to various diseases due to failure of one or more checkpoints.

Asymmetrical Membranes and Proteins

• Membrane Characteristics:

  • Membranes are asymmetrical, resulting in different environments on the cytosolic and extracellular sides.

  • Protein Compatibility:

  • Soluble proteins in the ER undergo modifications to be compatible with extracellular settings.

  • Membrane Protein Orientation:

  • Membrane proteins must maintain their orientation throughout their trafficking: differences in chemical environments necessitate this asymmetry for cellular function.

  • Asymmetry maintenance initiates in the ER and persists throughout the endomembrane system.

The notes provided ensure a comprehensive and thorough understanding of protein trafficking into the endoplasmic reticulum, encapsulating all essential details as extracted from the transcript.