ER - Golgi - Lysosomes

Department of Biological and Environmental Sciences
Cell & Molecular Biology
Dr. David A. Johnson
Biol 405 4 Credits Spring 2017 MWF 11:45-12:50 AM PH 204

<<< The ER, Golgi Apparatus, and Lysosomes: Sorting Proteins and Other Macromolecules >>>

These organelles are important in various aspects of the metabolism of proteins and other molecules, including their cellular or extracellular destiny.
The Endoplasmic Reticulum (ER) (Mother of All--well, not really--Organelles): This continuous network of membranous tubules and sacs run throughout the cell. Rough ER has ribosomes on the cytosolic surface. Transitional ER is involved with budding that sends vesicles to the Golgi. Both are important in protein processing. Smooth ER has no ribosomes attached and is involved in lipid metabolism. ER gives rise to Golgi, lysosomes, and new cell membrane.
ER Associated Proteins and Rough ER: Some proteins are targeted for the lumen of the ER or to be embedded in its membrane. (Their ultimate fate may be different--they may be transported elsewhere later.) Sending a protein into the ER is the process of translocating it across (or into) the ER membrane. All protein synthesis begins on ribosomes in the cytosol which are unattached to the ER (free ribosomes). Proteins that are destined to remain in the cytosol complete their synthesis on free ribosomes and are therefore released into the cytosol.
Cotranslational Translocation: Some proteins headed for the ER lumen enter the ER as they are being made (during translation).
Signal Sequences and Signal Recognition Particle (SRP): The synthesis of cotranslationally translocated proteins begins on free ribosomes. These proteins have a unique signal sequence that is near the N-terminus. The signal sequence is about a 20 amino acid sequence including a stretch of hydrophobic amino acids (α helix). A protein-RNA complex called signal recognition particle (SRP) recognizes and binds to the signal sequence and to the ribosome, halting translation.
SRP Receptor, Translocon, and Signal Peptidase: The mRNA-ribosome-polypeptide-SRP complex binds to a protein on the ER called the SRP receptor. SRP receptor binds to SRP and the ribosome binds to a protein complex next to the SRP receptor called a translocon. The translocon forms a channel into the interior of the ER. The binding of SRP receptor to SRP causes SRP to be released from its association with the signal sequence and the ribosome. (Research news) Translocation: With SRP gone, translation now resumes and the growing polypeptide is inserted into the channel in translocon. However, the signal sequence is retained within the translocon (binds to the wall of the channel) as the growing polypeptide is inserted into the ER lumen. Signal Peptidase: An enzyme associated with the translocon on the lumen side cleaves the signal sequence releasing the polypeptide into the lumen.
Posttranslational Translocation: While most ER lumen proteins are targeted for the ER by cotranslational translocation, some are made on free ribosome then translocated into the ER.
ER Membrane Proteins: Some ER proteins are not destined for the lumen of the ER but rather to be embedded into the ER membrane. These may have an internal signal sequence rather than an N-terminal one.
ER Membrane Proteins with an N-Terminal Signal Sequence and a Internal Stop-Transfer Sequence (Single Pass Membrane Proteins): Translocation of these proteins proceeds as described above in cotranslational translocation, but midway in the synthesis, a second sequence called a stop-transfer sequence (also an α helix) stops the translocation process. The stop-transfer sequence alters the translocon so that no further translocation occurs. Therefore, the rest of the polypeptide remains on the cytosolic side. The stop transfer α helix passes through the wall of the translocon and into the phospholipid bilayer. When translation is finished, this single pass membrane protein has its N-terminus on the lumen side and its C-terminus on the cytosolic side.
ER Membrane Proteins with Internal Signal Sequence(s) and/or Internal Stop-Transfer Sequence(s) (Single or Multiple Pass Membrane Proteins): Some membrane proteins have one or more internal signal sequences and stop-transfer sequences. The orientation of such single pass membrane proteins may be in either orientation (N-terminus outside or C-terminus outside--different signal sequences bind in opposite orientations). Some proteins have multiple internal signal sequences and stop-transfer sequences resulting in multiple-pass membrane proteins.
Protein Folding and Processing in the ER: Various changes occur to proteins in the ER. Chaperones and Folding: Polypeptides must assume the correct folding pattern in order to function properly. The correct folding of a protein is mediated by chaperones (they also are proteins--chaperones are abundant in the ER lumen). A completed polypeptide will assume the correct folding pattern spontaneously, however before translation is complete, it could assume an incorrect formation or it could aggregate with other partially made polypeptides. To prevent this, chaperones in the ER (and cytosol) bind to the nascent polypeptide and keep it from interacting with anything until the polypeptide is completely synthesized. (Chaperones bind to polypeptides destined for mitochondria then release them as they pass through the mitochondrial membranes. Chaperones on the inside of mitochondria bind until these polypeptides have completely entered.)
Cleavage: Many polypeptides have amino acids removed after translation. This may be simply the removal of the initial methionine or more extensive cleavage as occurs to preproinsulin in the pancreas. Preproinsulin has an N-terminal signal sequence (as above). Its removal produces proinsulin inside the ER. Then, in the ER lumen an internal amino acid sequence is removed and degraded and the resulting two polypeptides are joined by disulfide bridges to form insulin.
Protein Disulfide Isomerase and Disulfide Bridges: In the lumen, protein disulfide isomerase makes a covalent bond (S-S, disulfide bridge) between two cysteine residues. This enzyme makes and breaks these bonds over and over until the most stable configuration is formed. Disulfide bridges are found only in proteins that are to be secreted or are exterior membrane proteins, since the cytosol contains reducing agents that would break these S-S bonds.
Glycolsylation and Other Modification: Other chemical modifications occur in the ER lumen, such as the addition of oligosaccharides (glycosylation). External membrane proteins are glycosylated this way.
Lipids and Smooth ER: Most membrane lipids are synthesized in smooth ER. This includes phospholipids, glycolipids, and cholesterol. The synthesis of phospholipids occurs in the outer layer of the ER membrane bilayer. The enzyme flippase moves phospholipids to the inner membrane layer. Export from the ER, Transitional ER, and the Golgi Apparatus: Vesicles bud off of the ER and thereby carry ER lumen content and ER membrane components to the Golgi. These vesicle first fuse to the ER-Golgi intermediate compartment which gradually become cis-face cisternae of the Golgi. These gradually become the trans-face cisternae. From the trans-face, vesicles bud off to fuse with the cell membrane (secretion and cell membrane formation), or to fuse with endosomes/lysosomes. Golgi are abundant in secreting cell (exocytosis). Clathrin-Coated Vesicles: As vesicles bud off of the ER, they first become coated protein. One example of these coated vesicles is clathrin-coated vesicles. Through a complex series of reactions, the region of the ER to form a vesicle becomes coated with the protein clathrin in a network pattern. This binding stimulates budding because it distorts the membrane. After the vesicle has budded off, clathrin is removed. Membrane Fusion: A vesicle fuses with either the cell membrane or another membrane (e.g., endosome). This involves binding of v-SNARES on the vesicle with t-SNARES on the target membrane.




The Golgi Apparatus and Lysosomes: Lysosomes are small membranous sacs containing powerful hydrolytic enzymes. Lysosomal Acid Hyrdolases: Lysosomes contain lysosomal acid hydrolases (about 50 kinds) that can break down all cellular organic compounds. They only work in the acidic (ph ~5) environment of the lysosome. So, if one were to burst, the cell would be OK. Endocytosis and Endosomes: Extracellular material can be brought into a vesicle by endocytosis. Pinocytosis is endocytosis on a small scale and involves clathrin-coated endocytic vesicles. These vesicles f use with early endosomes (larger vesicles than the endocytic vesicles). Membrane is recycled to the cell membrane and early endosomes become late endosome. Hydrolases in the Golgi are carried by vesicles to the late endosomes and the material brought in from the outside by pinocytosis is digested. Lysosomes: Late endosomes mature into lysosomes with a high concentration of acid hydrolases. Phagocytosis and Lysosomes: Phagocytized materials (endocytosis on a large scale) enters the cell and a phagosome is formed. Lysosomes fuse with the phagosome digesting the phagocytized material. Autophagy and Lysosomes: Old organelles are surrounded by ER membrane and these sac becomes autophagosomes. Lysosomes fuse with these autophagosomes and the old organelles are digested.

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