Name: er glycoprotein folding checkpoint
Uploaded: 2023-04-30T10:07:45.000Z
Duration: 1 min 29 s
Channel: JoVE

In the ER lumen, an unfolded polypeptide has multiple precursor N-linked glycans attached to it.
These precursor glycans are branched oligosaccharides, each with three terminal glucose residues.
Two of these glucose residues are sequentially removed by the action of glucosidase I and glucosidase II, leaving behind a trimmed N-linked oligosaccharide with a single glucose.
The membrane-bound calnexin and soluble calreticulin are lectin chaperones, which specifically bind such monoglucosylated trimmed glycans.
The lectins then recruit other chaperones to assist in polypeptide folding.
Soon after, glucosidase II trims out the final glucose residue, creating non-glucosylated glycans on the polypeptide.
The UDP-glucose:glycoprotein glucosyltransferase 1, or UGGT1 enzyme, then assesses the protein for its folding accuracy.
If the folding is correct, the protein is allowed to exit the ER.&#160; Otherwise, UGGT1 adds a glucose molecule from UDP-glucose back to the glycan attached to the polypeptide.
The reglucosylation recycles the polypeptide for another round of folding by calnexin/calreticulin chaperones until the correct protein structure is achieved.

protein folding quality check in the rer

Protein Folding Quality Check in the RER

ER is the primary site for the maturation and folding of soluble and transmembrane secretory proteins. The calnexin cycle is a specific chaperone system that folds and assesses the confirmation of N-glycosylated proteins before they can exit the ER lumen. The primary players of this quality check pipeline are the lectins, ER-resident chaperones, and a glucosyl transferase enzyme. In case the calnexin system in the lumen fails to salvage a misfolded protein, it is transported to the cytoplasm for degradation.
Lectin Chaperone System: The Glycoprotein Binding Proteins
The lectin chaperones &#8212; calnexin, and calreticulin are distinguished from other ER chaperones due to their ability to bind N-linked glycans. The N-linked glycans are abundantly present on most polypeptide chains entering the ER lumen, and the lectins utilize this modification for proofreading the accuracy of protein folding. Calnexin and calreticulin, both have very similar functional domains. They primarily bind mono-glucosylated N-linked glycans using their glycan-binding lectin domain, and use the P-domain on their flexible arm to interact with other ER chaperones.
Lectin Binding ER Chaperones: The Accessory Helpers
Three functionally distinct ER chaperones &#8212; Erp57, cyclophilin B, and Erp29, assist the calnexin/calreticulin proteins. ERp57 is a protein disulfide isomerase that catalyzes the oxidation and isomerization of disulfide bonds. While cyclophilin B rearranges and corrects the disulfide bonds, ERp29 is a general chaperone that aids peptide folding.
UDP-glucose:glycoprotein Glucosyltransferase 1: The Proofreader
UDP-glucose:glycoprotein glucosyltransferase 1, or UGGT1, a protein folding sensor, binds polypeptide and acts as the single glycoprotein folding quality control checkpoint. It marks incorrectly folded glycoproteins to be retained in the ER by selective reglucosylation. The N-terminal of UGGT acts as the misfold sensor, whereas the C-terminal harbors the reglucosylating glucosyltransferase.

ER is the primary site for the maturation and folding of soluble and transmembrane secretory proteins. The calnexin cycle is a specific chaperone system ...

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JoVE Core

Cell Biology

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

transmembrane-transport-in-endoplasmic-reticulum-and-peroxisomes

KEY TERMS AND CONCEPTS

er glycoprotein folding checkpoint

The endoplasmic reticulum, or ER, is the main site where soluble and transmembrane secretory proteins mature and fold. A key part of this process is the calnexin cycle, a chaperone system that folds and checks N-glycosylated proteins before they leave the ER lumen. If this system cannot rescue a misfolded protein, the protein is sent to the cytoplasm for degradation.

Calnexin and calreticulin are lectin chaperones. Lectins are glycoprotein-binding proteins, and these two are unique because they bind N-linked glycans on many polypeptide chains entering the ER lumen. They use this sugar tag to help proofread whether a protein has folded correctly. Both proteins have similar functional domains. Their lectin domain binds mono-glucosylated N-linked glycans, while the P-domain on a flexible arm helps them interact with other ER chaperones.

Several ER chaperones assist this folding system. ERp57 is a protein disulfide isomerase that helps oxidize and isomerize disulfide bonds. Cyclophilin B rearranges and corrects disulfide bonds. ERp29 acts as a general chaperone that supports peptide folding.

UGGT1, or UDP-glucose:glycoprotein glucosyltransferase 1, serves as the folding quality control checkpoint. It is a protein folding sensor that binds polypeptides and keeps incorrectly folded glycoproteins in the ER by selective reglucosylation. Its N-terminal region acts as the misfold sensor, and its C-terminal region contains the reglucosylating glucosyltransferase.

ER Glycoprotein Folding Checkpoint

the endoplasmic reticulum

ER Membranes, Microsomes, and Cell Support

The endoplasmic reticulum, or ER, is a major membrane network in eukaryotic cells. It makes up more than half of a cell’s membranes and about 10% of total cell volume. The ER also serves as a main site for protein and lipid synthesis for organelles such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. The ER was also the last major cell organelle to be discovered. In 1954, Keith Porter and George Palade produced the first high-resolution electron microscope...

smooth endoplasmic reticulum

Smooth ER in Lipids, Sugar, and Detox

Smooth endoplasmic reticulum, or smooth ER, is a cell organelle with specialized jobs in both animal and plant cells. It is often linked to the tubular shape of the endoplasmic reticulum. Its main role is to support processes that need the right internal conditions inside the cell. Smooth ER is an important site for making steroid hormones and lipids. These lipids include phospholipids and triglycerides. It also makes major membrane lipids such as phosphatidylcholine, phosphatidylethanolamine,...

role of er in the secretory pathway

ER Quality Control in Protein Secretion

The endoplasmic reticulum (ER) helps control protein secretion in eukaryotic cells. It acts as the first checkpoint in the secretory pathway, where many proteins are folded, modified, and tested for quality before they move on. The secretory pathway links membrane-bound compartments inside the cell and also connects cells to the outside environment. About one-third of the proteins made in a cell travel this route. These proteins move in membrane-bound vesicles between the ER, the ER-Golgi...

directing proteins to the rough endoplasmic reticulum

directing-proteins-to-the-rough-endoplasmic-reticulum

How SRP Targets Proteins to Rough ER

Proteins made in the cytosol often need a signal sequence to reach the rough endoplasmic reticulum (ER). These organelle-specific signals act like address tags and guide proteins to places such as the ER, mitochondria, and peroxisomes. Proteins sent to the ER may later move in vesicles through the Golgi complex to other organelles or leave the cell. The rough ER makes soluble proteins that can be sent to lysosomes or secreted outside the cell. It also makes transmembrane proteins, which become...

protein translocation machinery on the er membrane

protein-translocation-machinery-on-the-er-membrane

ER Membrane Translocon and Protein Entry

The translocon complex on the ER membrane acts as the main gateway for the protein secretory pathway. It moves new peptides into the ER lumen and also helps insert proteins into the ER membrane. In eukaryotes, the core of this complex is the Sec61 channel. Sec61 is a heterotrimer made of Sec61α, Sec61β, and Sec61γ, and it is the largest part of the translocon. In higher eukaryotes such as mammals, the channel is capped when it is resting. This helps prevent ions such as calcium from leaking...

cotranslational protein translocation

Sec61 and SRP in Protein Targeting

Sec61 and SRP help move newly made proteins across membranes during cotranslational translocation. This process is ancient and has been found in bacteria, archaebacteria, and eukaryotes. The machinery is conserved across these groups, which shows how important it is for cells. During cotranslational translocation, the Sec61 channel works with the signal recognition particle, or SRP, the SRP receptor, or SR, and ribosomes. SRP binds the ribosome through the polypeptide exit tunnel in the large...

post-translational translocation of proteins to the rer

post-translational-translocation-of-proteins-to-the-rer

Sec62-Sec63 Pathway for ER Protein Import

Post-translational translocation moves some proteins from the cytosol into the endoplasmic reticulum (ER) after they are made. These proteins still use the Sec translocon complex to cross the ER membrane and enter the ER lumen. During this process, Hsp40 and Hsp70 chaperones bind the new protein in the cytosol and help stop it from folding too soon. That chaperone binding also keeps the signal sequence exposed so it can contact the Sec61 channel. The ER signal sequence has features that help...

insertion of single-pass transmembrane proteins in the rer

insertion-of-single-pass-transmembrane-proteins-in-the-rer

Single-pass Membrane Protein Topology in the ER

Single-pass membrane protein topology in the ER explains how certain proteins are built into a cell membrane. Integral membrane proteins are attached to the lipid bilayer of a cell organelle or membrane. They may span the membrane as transmembrane proteins, or they may sit on one side only as monotopic proteins. Transmembrane proteins have both membrane-spanning and extra-membrane domains. Their transmembrane regions usually contain 20-25 hydrophobic amino acids arranged in a helical shape.

insertion of multi-pass transmembrane proteins in the rer

insertion-of-multi-pass-transmembrane-proteins-in-the-rer

How Multipass Proteins Enter the ER Membrane

Multipass transmembrane proteins enter the rough ER membrane through a stepwise insertion process. These proteins are type IV integral membrane proteins, and they use multiple topogenic sequences to set their final position in the membrane. They can stay in the ER or later move to the Golgi complex, lysosome, or cell membrane. Most multipass proteins do not have a cleavable signal sequence. Instead, their first hydrophobic segment, also called a transmembrane domain, acts as the ER signal...

tail-anchoring of proteins in the er membrane

tail-anchoring-of-proteins-in-the-er-membrane

ER Membrane Targeting of Tail-Anchored Proteins

Tail-anchored proteins are a group of membrane proteins that reach the endoplasmic reticulum, or ER, after they are made. They make up about 3% to 5% of membrane proteins in eukaryotic cells. These proteins have one transmembrane domain near the C-terminal end, which is the last part of the protein chain. Because the transmembrane segment is so close to the end, the signal recognition particle, or SRP, cannot bring these proteins to the ER for cotranslational insertion. Cotranslational...

gpi anchoring of proteins in the er membrane

gpi-anchoring-of-proteins-in-the-er-membrane

How GPI-Linked Proteins Reach the Membrane

GPI-linked proteins are made through a post-translational modification that helps place them on the outer side of the plasma membrane in eukaryotic cells. The GPI, or glycosylphosphatidylinositol, anchor is reversible and is built through 11 enzyme-driven steps. It includes a hydrophobic part and a hydrophilic part. The hydrophobic region contains phosphatidylinositol. The hydrophilic region includes phosphoethanolamine, glucosamine, mannose residues, and phosphate groups. Phosphoethanolamine...

protein modifications in the rer

Rough ER Protein Folding Changes

Rough ER protein folding changes help secretory and transmembrane proteins mature inside the ER lumen. These changes support folding and help stabilize the protein’s final three-dimensional shape. They happen at different stages as the protein folds. The main rough ER modifications fall into four groups: glycosylation, disulfide bond formation, assembly of protein subunits, and specific proteolytic cleavages, such as removal of signal sequences. N-linked glycosylation is the most common. In...

The endoplasmic reticulum, or ER, is the main site where soluble and transmembrane secretory proteins mature and fold. A key part of this process is the calnexin cycle, a chaperone system that folds and checks N-glycosylated proteins before they leave the ER lumen. If this system cannot rescue a misfolded protein, the protein is sent to the cytoplasm for degradation. Calnexin and calreticulin are lectin chaperones. Lectins are glycoprotein-binding proteins, and these two are unique because...

export of misfolded proteins out of the er

export-of-misfolded-proteins-out-of-the-er

How the ER Clears Misfolded Proteins

The endoplasmic reticulum (ER) checks secretory and membrane proteins after they fold. Correctly folded proteins move through the calnexin cycle and continue to their final destination. Misfolded proteins are kept in the ER lumen, where ER chaperones try to unfold and refold them. When refolding fails, the protein becomes terminally misfolded. The ER then sends it to the cytosol through ER-associated degradation, or ERAD, for destruction. In this pathway, Hrd1 acts as both a ubiquitin ligase...

the unfolded protein response

ER Stress Control by the UPR

The endoplasmic reticulum, or ER, is a major site of protein synthesis in the cell. It also has strong quality control systems for protein folding and for breaking down misfolded proteins that cannot be fixed. Under normal conditions, some misfolded proteins are sent from the ER to the cytoplasm through ER-associated degradation, or ERAD. If ERAD cannot keep up, the cell turns on the unfolded protein response, or UPR. This response helps the cell adjust its protein folding pathways and reduce...

regulation of the unfolded protein response

regulation-of-the-unfolded-protein-response

IRE1 Signaling in ER Stress

IRE1 signaling helps cells respond to stress in the endoplasmic reticulum, or ER. IRE1, short for inositol-requiring kinase one, is the most conserved eukaryotic unfolded protein response receptor. It is a type I transmembrane protein kinase receptor with a site-specific RNase, or RNA-cutting, activity. The way misfolded proteins bind to the N-terminal domain of IRE1 is still not fully clear. Three activation models have been proposed: direct, indirect, and allosteric. Once a misfolded protein...

synthesis of phosphatidylcholine in the er membrane

synthesis-of-phosphatidylcholine-in-the-er-membrane

Phosphatidylcholine’s Roles in Cell Membranes

Phosphatidylcholine is a major glycerophospholipid in cell membranes. The endoplasmic reticulum, or ER, makes lipids that help build membranes and support cell functions such as energy storage and signaling. The lipid-making machinery sits in the ER membrane and gathers its reactants from the cytosol, the fluid inside the cell. After phosphatidylcholine and other lipids are made, the cell moves them to other organelles through the secretory pathway and through ER contact sites. Energy-rich...

assembly of the lipid bilayer in the er

How the ER Balances Membrane Lipids

The endoplasmic reticulum (ER) helps build and balance membrane lipids in cells. Biological membranes are not just barriers. They also keep cells and organelles stable and support communication between and within cells. Many membranes are made largely of phospholipids, which are a major type of membrane lipid. These lipids are not spread evenly. They differ from one organelle to another and even between the two layers, or leaflets, of a membrane. The ER is the starting point of the secretory...

peroxisomes

Peroxisome Functions in Cells

Peroxisomes are specialized organelles found in fungi, plant, and animal cells. Their number, size, shape, and activity can change with the tissue type and the cell’s nutritional state. Cells with active lipid metabolism, such as adipocytes, neurons, and hepatocytes, often contain more peroxisomes than other cells. Peroxisomes help break down complex organic molecules. They also synthesize certain macromolecules and take part in redox signaling, which is a way cells use oxidation and reduction...

protein import into the peroxisomes

How Peroxisomes Receive Their Proteins

Peroxisomes receive many of their proteins from the cytoplasm and the endoplasmic reticulum. These membrane-bound organelles oxidize organic molecules by transferring hydrogen atoms to oxygen. This process produces hydrogen peroxide, which peroxisomes then break down into water and oxygen. Peroxisomes do not have the genetic machinery to make their own proteins. Most peroxisomal membrane, lumenal, and transmembrane proteins are made in the cytoplasm or the ER and then moved to the peroxisome.

key terms and concepts

Learn how calnexin, calreticulin, ER chaperones, and UGGT1 check N-glycosylated proteins in the ER before they leave the lumen.

ER Glycoprotein Folding Checkpoint | JoVE K12

cell biology

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ER Glycoprotein Folding Checkpoint

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