BL 424 Chapter 10: Protein sorting and transport: The endoplasmic reticulum, golgi apparatus, and lysosomes

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1 BL 424 Chapter 10: Protein sorting and transport: The endoplasmic reticulum, golgi apparatus, and lysosomes Student Learning Outcomes: Proteins are the active players in most cell processes. They need to get to the correct compartments in the cell and with the correct modifications. 1*. Explain the process of sorting and transport of proteins in cells, especially the initial sorting between proteins synthesized on free (cytosolic) ribosomes and those bound to the ER (rough ER). 2*. Describe the signals on proteins that identify their destinations: amino acid sequences on new proteins, carbohydrate chains, mannose phosphate, lipids 3*. Describe the diverse protein complexes that read the signals and assist delivery of proteins to the correct place: SRP particles, signal peptidases, chaperones, vesicle receptors, SNARE proteins 4*. State concisely the steps taken by different proteins: destined for secretion, for incorporation into a lysosome; indicate the compartments it traverses, and the protein complexes involved. 5. Appreciate the experimental basis behind these concepts, and the pathological (disease) states that can derive from malfunctions. Important Figures: 2, 3**, 5, 6, 8*, 9*, 10, 11*, 12, 13, 14*, 15, 16, 17*, 20*, 21, 23*, 25*, 27*, 31*, 32, 35, 37, 39*, 43, 44 Important Tables: 1 1*. The endoplasmic reticulum (ER) is the first branch point for protein sorting. The ER is membrane-enclosed tubules and sacs extending from the nuclear membrane. The rough ER has ribosomes associated; The smooth ER does not have ribosomes. Secreted proteins travel from the rough ER -> Golgi -> secretory vesicles -> cell membrane (Fig. 10.2) Experiments with pancreatic cells with radioactively labeled aa Brief label, then chase with unlabeled aa *Proteins destined for nucleus, mitochondria, chloroplasts or peroxisomes are synthesized on free ribosomes in the cytosol (Fig **). Proteins destined for secretory vesicles, plasmia membrane, lysosomes, endosomes or to be retained in the ER or Golgi are synthesized on membrane-bound ribosomes, then transferred into the lumen of the rough ER, either as translation proceeds (co-translational) (most mammals) or after translation (post-translational)(yeast, some mammals).

2 All protein synthesis begins in the cytosol on a ribosome. Proteins destined for secretion, lysosome or to be retained in the ER or Golgi then move to the ER. Signal sequences on the N-terminus of a protein target the ribosomes to the ER (Fig. 10.5; Fig. 8.7). Signal sequences are often hydrophobic (Fig. 10.6) Ex. Of growth hormone For co-translational targeting of secretory proteins, Signal sequences bind signal recognition particles (SRP) which are composed of RNA and protein (Fig. 10.7). Protein-ribosome-SRP binds SRP receptor on ER (Fig. 10.8). SRP is released; Signal sequence is inserted in the translocon channel; Protein synthesis translocates chain across membrane; Signal sequence is cleaved by signal peptidase (in lumen) to release the final polypeptide in the ER lumen. Post-translational targeting of proteins to ER lumen uses cystolic chaperones (Hsp70 proteins) to protect the nascent polypeptides; Signal sequence is recognized by the Sec62/63 complex that is associated with a translocon; (Fig. 10.9). Hsp70 BiP protein chaperone inside ER assists with translocation Integral membrane proteins (those destined to be inserted in plasma membrane of ER, Golgi, endosome or lysosome) are initially targeted and inserted into ER membrane, but then are anchored by membrane-spanning α-helices that stop transfer of the growing chain across membrane. [(Figs. 9.10, 12-14) show proteins with different orientations, different numbers of membrane-spanning helices. Internal signal sequences are present for proteins with multiple membrane spans. These proteins move through compartments but are always membrane-bound.] *** Topology of the secretory pathway (Fig *): A protein in the lumen of the ER or Golgi is topologically equivalent to the exterior of the cell. Therefore, portions of a polypeptide translocated into the ER are exposed on the cell surface after transport to plasma membrane

3 Protein folding and processing for secretory proteins occurs in the ER: Cleavage of proteins may occur (as from signal sequence) It involves chaperones (Hsp70, BiP) (Fig ) formation of disulfide bonds by PDI (see Fig. 8.24), N-linked glycosylation on Asn residue helps proteins fold (Fig , ) Addition of GPI anchors (Fig , 8.35): these proteins will be exposed on outside of cell Quality control checks in the ER lumen: Improperly folded proteins are degraded. The glycoprotein chaperone calreticulin checks nascent glycoproteins (Fig ), sends to sensor; improper proteins are retro-translocated, degraded Chaperone BiP assists with folding in ER; In stressed cell, too many unfolded proteins lead to The Unfolded protein response: with Inhibition of protein synthesis, increased chaperones. Smooth ER is the site of lipid synthesis. Smooth ER is abundant in cells active in lipid synthesis. 3 main lipids are phospholipids, glycolipids and cholesterol. Phospholipids are synthesized in cytosolic side of ER (from water-soluble precursors), and then translocated across membrane by flippase enzyme (Figs. 20, 21). Cholesterol and ceramide are made in the ER (Fig. 22) Cholesterol is basis for other steroid hormones Ceramide is converted to sphingomyelin or glycolipid in the Golgi. Vesicles transport proteins and lipids from the ER to the Golgi, fusing to deposit cargo. Proteins first go to ERGIC (Fig *). (ER-Golgi intermediate compartment) Different targeting sequences mediate packaging, transport to Golgi, including di-acidic aa, hydrophobic aa or GPI anchors (Fig ). Resident ER proteins like BiP have special aa sequences at COOH end that signal their return to ER: ex. A KDEL sequence (or KKXX) on a protein is recognized by receptor for return to ER (Fig *).

4 2*. The Golgi apparatus functions in protein processing and sorting as well as in synthesis of lipids and polysaccharides. Structure: stacks of membranes: (Fig *) Cis stack oriented towards ER; Medial and trans stacks in middle; Trans-Golgi network is most distal. Vesicles carry proteins from one stack to another, and then to final destination as endosome, lysosome, plasma membrane or secreted to exterior. Different proteins get different modifications: Glycosylation: (recall also Chapter 8): Glycosyltransferases add sugars; Glycosidases remove sugars. N-linked oligosaccharides that were added to Asn residues of proteins in the ER (9 mannoses, 2 N-acetylglucosamines) are further modified in the Golgi (Fig ). Mostly for secreted, plasma membrane proteins: orderly process of removal and addition, but varies with cell type and protein. Lysosomal proteins get phosphorylation of mannose: Mannose-6-phosphate is formed by addition of N-acetylglucosamine phosphate, and then removal of the N-acetylglucosamine. (Fig ). Specific mannose-6-phosphate receptor in the trans Golgi network will direct transfer. The enzyme adding the mannose-6-phosphate recognizes particular shape of folded protein, signal patch, rather than linear aa sequence. O-linked sugars are added in the Golgi to OH groups of Ser and Thr residues. Lipid and polysaccharide metabolism in the Golgi. The Golgi is site for synthesis of synthesis of Glycolipids and sphingomyelin from ceramide; Ceramide was made on smooth ER (Fig ). complex polysaccharides of plant cell walls are made in the Golgi (but not cellulose). (Chapter 14).

5 Protein sorting and export from the Golgi apparatus. Proteins are sorted in the trans Golgi network, packaged into transport vesicles for destinations: (Fig *): secretion (regulated or constitutive), plasma membrane, lysosomes or vacuoles(yeast or plants); Constitutive secretion is default pathway. Polarized cells such as intestinal epithelial cells have proteins targeted to apical or basolateral domains of the plasma membrane (Fig ). 3*. Mechanisms of vesicular transport. Vesicles have important role in traffic of molecules in the secretory path. Vesicles are important in uptake of materials from cell surface (Chapt. 13). Selectivity, specificity of packaging and transport is critical for function. Experimental approaches to understanding transport include studies of packaging, budding and fusion, using model systems of: yeast mutants (defective for secretion or transport), reconstituted cell-free systems, synaptic vesicles (ex. acetylcholine neurotransmitter) and GFP-fusion proteins. Cargo selection, coat proteins and vesicle budding Most vesicles carrying secretory proteins have Coats of cytosolic proteins, coated vesicles. Cargo proteins are sorted into vesicles, cargo-containing bud forms, and the vesicle moves to target membrane and fuses to release cargo. (Fig , 10.35). 3 Types of coated vesicles for different destinations: Clathrin-coated, non-clathrin coated (COPI, COPII) Formation of coated vesicles is regulated by 3 families of GTP binding proteins: (Fig ) (ARF1-3, Sar1, and the Rab family); They regulate binding of adaptor proteins that will bind the 3 types of coat proteins. Clathrin- coated vesicles (Fig ) carry proteins for endosomes and lysosomes, and for endocytosis (see Chapter 13). Mannose-6-phosphate on protein cargo binds the membrane mannose-6-phosphate receptor, which binds an adapter, ARF and the clathrin. Non-clathrin-coat types of vesicles are: COPII (coat protein II): Carry secretory proteins from ER to ERGIC. COPI carry cargoes through Golgi, and from Golgi to ER.

6 Vesicle fusion involves recognition between vesicle and target membrane, then fusion and delivery of contents. SNARE hypothesis: recognition involves specific pairs of transmembrane proteins (SNAREs), on vesicle and target. Binding of v-snare to t-snare provides energy to bring two bilayers close together for fusion. Docking and tethering of vesicle to membrane are mediated by protein complexes, and with more GTP-binding proteins of the Rab family. (Table 1, Fig , 10.39). Disassembly requires ATP and SNAP proteins Exocyst assembly (Fig ) Evidence comes from experiments with Yeast secretion (exocytosis) Synaptic vesicle (neurons) release from vertebrates. *Lysosomes are membrane-enclosed organelles with many digestive enzymes, or acid hydrolases that degrade macromolecules. Extracellular molecules taken up by endocytosis are transported to endosomes, which mature to lysosomes when vesicles with acid hydrolase proteins are delivered from the Golgi, and a proton gradient is established across the membrane (Figs , 43). At least 30 human diseases are due to defects in Lysosomal enzymes (lysosomal storage diseases) Ex. Tay Sachs disease; Gaucher s disease Products build up, impair function of cells. Phagocytosis and Autophagy involve lysosomes degrading large particles and recycling organelles (Fig ). Review questions at end of chapter are all. ** Diagram an amino acid sequence for a protein that would be secreted, for one that is destined for the nucleus, and for a protein that would be a membrane-protein inserted in the ER.