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ProteinTransport

Page history last edited by Derek Weber 13 years, 7 months ago

The Endomembrane System

Rather than review all parts of chapter 5, I have decided to focus on one aspect of the chapter: the endomembrane system. The interior of a eukaryotic cell is divided into many membrane bound compartments that make up the endomembrane system (figure below).

As you can see, the endomembrane system contains a series of membrane bound compartments that include the: endoplasmic recticulum, the Golgi apparatus, the lysosome, and the plasma membrane. Small bubbles of membrane called vesicles also play a key role in the endomembrane system. The overall function of the endomembrane system is to produce and coordinate the movement of macromolecules like lipids and proteins throughout the cell. In some cases, parts of the endomembrane system are involved in processing as well. The following sections will address specific pathways within the endomembrane system. Please take note of the roles each compartment plays in the process.

 

A. Protein Synthesis

As we learned earlier, proteins are synthesized by ribosomes and play a critical role in carrying out important cellular processes. An important aspect of protein function is cellular location. Proteins are classified into four classes based on their final destination:

Class I: function in the cytoplasm to aid in metabolism

Class II: act as enzymes that function within different cellular compartments.

Class III: aid in transport into and out of the cell as membrane-bound proteins.

Class IV: work outside of the cell in which they were synthesized as secreted proteins.

 

Class I proteins that function solely in the cytoplasm are produced by "free ribosomes". These unattached ribosomes synthesize and deposit proteins directly in the cytoplasm. Proteins falling into one of the final three classes (II,III,IV) are produced by "bound" ribosomes. These ribosomes complex with the endoplasmic recticulum,synthesize and deposit proteins into the rough ER.

 

Overview of protein-trafficking in the cell. Class I proteins (cytosolic proteins) are produced on free ribosomes and are deposited in the cytoplasm. Some proteins have organelle-specific sequences (steps 3-6 right). These proteins are produced in the cytoplasm but are eventually deposited into their final organelles of interest (for example, proteins are required in the mitochondria, chloroplast, peroxisome, and nucleus to ensure their proper function within the cell). Class II-IV proteins are bound by ribosomes in the cytoplasm. However, the presence of an ER-sequence directs the ribosome to the ER (rough ER) where the protein is deposited and sent to its final destination via the endomembrane system. Please click the link above to view an animation of this process.

The following is an outline of the process of producing Class II-IV proteins.

Step 1: mRNA for the corresponding protein of interest is produced in the nucleus. Genes encode for products (like protein) that allow a cell to adapt to its environment. The DNA sequence that corresponds to the gene of interest is copied into mRNA via transcription. Many copies of mRNA can be produced from a single gene. This mRNA is then shuttled to the cytoplasm via the nuclear pores.

Step 2: Ribosomes bind to mRNA and begin translation. mRNA contains sequences that function as ribosome-binding sites (RBS). These sites are recognized by the small ribosomal subunit and the process of translation (protein synthesis) begins. As the peptide chain elongates, it exits the ribosome and becomes exposed to the cytoplasm.

Step 3: Binding of signal recognition particle (SRP) to the growing polypeptide chain. Proteins belonging to classes II-IV contain a 16-to 30-amino acid ER signal sequence that directs the ribosome to the ER membrane. This signal sequence is located in the N-terminus of the nascent peptide chain. This signal sequence demonstrates a general pattern of one of more positively charged amino acids adjacent to a continuous stretch of 6-12 hydrophobic residues. This signal sequence is not well conserved from a standpoint of the number of positively-charged residues and hydrophobic residues, but all secretory proteins demonstrate this general pattern. The hydrophobic residues are of critical importance. Mutations or deletions within this signal sequence results in the deposition of the protein in the cytoplasm, rather than the rough ER. Based on these studies, it is clear that this stretch of amino acids is critical to proper protein trafficking. The question must then be asked: What targets the ER-sequence? There are two proteins within a cell (called the signal recognition particle (SRP) and its receptor) that target the peptide to the membrane of the ER. The SRP protein binds to the conserved hydrophobic sequences found in the ER-targeting sequence and brings the ribosome to the membrane surface of the ER.

 

Structure of SRP. The protein pictured is a bacterial protein called Ffh. This protein is homologous to the portion of SRP that binds to the ER-sequence. In this surface model, you can observe the many hydrophobic amino acids (in blue) that line the binding cleft required to bind the ER-sequence. The make-up of this cleft coincides with the requirement for hydrophobic amino acids in the ER signal (adapted from R.J. Keenan et al., 1998, Cell 94: 181 and obtained from Molecular Cell Biology by Lodish et al).

Step 5: Completion of protein synthesis. Once the ribosome is bound to the ER-membrane, the SRP particle is released and the synthesis of the nascent chain is completed. An important side-note is the fate of the ER-sequence. This sequence does not play a role in the final 3-D structure of the protein and is cleaved from the rest of the chain before completion of the chain.

 

For proteins destined to become membrane-bound, this step differs. For these proteins, insertion into the membrane begins in the rough ER where they remain embedded until they reach there final destination. Membrane-bound proteins may contain one or more α-helices containing 20-25 hydrophobic amino acids. The amino acids are crucial in allowing the protein to become anchored in the hydrophobic core of a membrane. In some cases, membrane-bound proteins contain a single transmembrane domain, while other proteins contain seven transmembrane domains (figure below). It is not important that you memorize the different types of membrane topologies; the most important part of this is understanding the membrane-bound proteins found in the ER, Golgi, lysosome, and even the plasma membrane are synthesized and inserted in the rough ER membrane and then taken to their final destination while remaining membrane bound. Also, as we begin to learn more about the specific transmembrane domain sequences found in the different types of topologies, scientists often can predict the location and function of an unknown protein based on its amino acid sequence.

Focus on research:

How did scientists discover that proteins belonging to classes II-IV are deposited into the rough ER?

First, they used a sulfur isotope (35S) to label all newly synthesized protein. Cells were only exposed to the isotopes for short periods of time so that only newly synthesized proteins are labeled. Thought question: Based on what we learned about protein and nucleic acids, why did the scientists choose sulfur and not nitrogen isotopes to label protein? The treated cells are then homogenized (broken up) shearing the rough ER into small membrane particles called microsomes and these microsomes are purified. They then treated the microsomes with detergent (detergent disrupts the integrity of the microsomes) and added the enzyme protease (this enzyme cleaves peptide bonds and degrades peptide chains). They found that only the proteins not protected by a microsome were digested by the protease. The microsomes not treated with detergent contained intact proteins suggesting that proteins enter the ER after they are completed being synthesized.

 

Step 6: Modification of the protein chain within the rough ER. Before leaving the rough ER, proteins undergo four main modifications:

1. addition of carbohydrates

2. formation of any disulfide linkages

3. proper folding of the protein as well as assembly if it is a multi-subunit protein

4. cleavage of the chain to aid in folding

What is the purpose of such modifications? These modification generally promote proper folding and increase the stability of a proteins. The addition of a carbohydrate to a protein forms a glycoprotein. Glycoproteins often serve as cell-surface receptors important in identification of a cell type. These are also used in cell-to-cell adhesion and communication. Different cell types have different glycoproteins on their surface which make them distinguishable at the cellular level.

 

Clinical Application. α1-antitrypsin is a protein normally produced by macrophages and hepatocytes and secreted into the blood. α1-antitrypsin inhibits a blood protein called elastase, a protein degrading enzyme. This inhibition of elastase in the blood protects the fine tissue in the lung from damage. In one hereditary form of emphysema, there is a mutation that prevents antitrypsin from folding properly in the rough ER. This proteins builds up in the cell, preventing secretion. This prevents the fine tissue from absorbing oxygen efficiently, leading to the symptoms associated by emphysema.

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