11 Protein Sorting and Transport: The Endoplasmic Reticulum, Golgi Apparatus, and Lysosomes.

11 Protein Sorting and Transport: The Endoplasmic Reticulum, Golgi Apparatus, and Lysosomes

11 Protein Sorting and Transport
The Endoplasmic Reticulum The Golgi Apparatus The Mechanism of Vesicular Transport Lysosomes

Introduction In addition to a nucleus, eukaryotic cells have membrane-enclosed organelles in the cytoplasm. Subdivision of the cytoplasm allows these cells to function efficiently in spite of their large size—at least a thousand times the volume of bacteria.

Introduction Sorting and targeting of proteins to appropriate destinations are important tasks. The endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes are involved in protein processing and are connected by vesicular transport.

The Endoplasmic Reticulum
The endoplasmic reticulum (ER) is a network of membrane-enclosed tubules and sacs (cisternae), extending from the nuclear membrane throughout the cytoplasm. The membrane is continuous and is the largest organelle of most eukaryotic cells.

Figure 11.1 The endoplasmic reticulum (ER) (Part 1)

Figure 11.1 The endoplasmic reticulum (ER) (Part 2)

The ER has two domains that perform different functions: Rough ER is covered by ribosomes on the outer surface. Smooth ER has no ribosomes and is involved in lipid metabolism.

The role of the ER in protein processing and sorting was first demonstrated by Palade and colleagues in the 1960s. They studied pancreatic acinar cells that secrete digestive enzymes into the small intestine. Newly synthesized proteins were labeled with radioisotopes.

Location of the radiolabeled proteins was then determined by autoradiography. After labeling, incubation with non- labeled amino acids for different lengths of time (called a “chase”) allowed them to track the labeled proteins through the ER, Golgi apparatus, secretory vesicles, and then outside the cell.

These experiments defined the secretory pathway: Rough ER → Golgi → secretory vesicles → cell exterior Further studies showed a similar pathway for non-secreted proteins.

Figure 11.2 The secretory pathway

In eukaryotic cells, initial sorting takes place while translation is in progress. Proteins synthesized on free ribosomes stay in the cytosol or are transported to the nucleus and other organelles. Proteins synthesized on membrane- bound ribosomes are translocated directly into the ER.

Figure 11.3 Overview of protein sorting (Part 1)

Figure 11.3 Overview of protein sorting (Part 2)

Ribosomes are targeted to the ER by a signal sequence at the amino terminus. The signal is removed when the growing polypeptide chain enters the ER.

The role of signal sequences was determined by in vitro preparations of rough ER. When cells are disrupted, the ER breaks up into small vesicles called microsomes.

Figure 11.4 Isolation of rough ER

Blobel and Sabatini (1975) found that translation of secretory proteins on free ribosomes retained the signal sequences and were slightly larger. When microsomes were added, the growing polypeptides were incorporated into the microsomes and the signal sequences were removed.

Figure 11.5 Incorporation of secretory proteins into microsomes (Part 1)

Figure 11.5 Incorporation of secretory proteins into microsomes (Part 2)

Key Experiment, Ch. 11, p. 402 (2)

Signal sequences (about 20 amino acids) include a stretch of hydrophobic residues, and are usually located at the amino terminus of the polypeptide chain.

Figure 11.6 The signal sequence of growth hormone

Cotranslational targeting: As they emerge from the ribosome, signal sequences are bound by a signal recognition particle (SRP). SRPs consist of six polypeptides and a small cytoplasmic RNA (SRP RNA).

The SRP binds the ribosome and the signal sequence, inhibiting further translation. The entire complex binds to an SRP receptor on the rough ER membrane.

The SRP is then released, and the ribosome binds to a membrane channel or translocon. The signal sequence is inserted into the translocon, and translation resumes. The signal sequence is cleaved by signal peptidase and released into the ER lumen.

Figure 11.7 Cotranslational targeting of secretory proteins to the ER

Yeast and mammal translocons are complexes of three transmembrane proteins (Sec61 proteins). They are closely related to plasma membrane proteins that translocate secreted polypeptides in bacteria.

Figure 11.8 Structure of the translocon (Part 1)

Figure 11.8 Structure of the translocon (Part 2)

Insertion of the signal sequence opens the translocon by moving a plug away from the channel. The growing polypeptide is transferred through the translocon as translation proceeds. The signal sequence is cleaved by signal peptidase and the polypeptide is released into the ER lumen.

In posttranslation translocation (more common in yeast), polypeptides are targeted to the ER when translation is complete. The signal sequences are recognized by different receptors on the translocon. Hsp70 and Hsp40 chaperones keep the polypeptide chains unfolded so they can enter the translocon. 33

Another Hsp70 chaperone in the ER (BiP) acts as a ratchet to pull the polypeptide chain through the channel and into the ER.

Figure 11.9 Posttranslational translocation of proteins into the ER

Proteins destined for incorporation into membranes are initially inserted into the ER membrane. They are transported along the secretory pathway as membrane components rather than as soluble proteins.

The membrane-spanning regions of integral membrane proteins are usually α helical regions with hydrophobic amino acids. Orientations vary—the amino (N) or the carboxy (C) terminus is on the cytosolic side; some proteins have multiple membrane-spanning regions.

Figure 11.10 Orientations of membrane proteins

The lumen of the ER is topologically equivalent to the exterior of the cell. Domains of membrane proteins that are exposed on the cell surface correspond to regions of polypeptide chains that are translocated into the ER lumen.

Figure 11.11 Topology of the secretory pathway

Many proteins are inserted directly into the ER membrane by internal transmembrane sequences. These sequences are recognized by SRP, but not cleaved by signal peptidase.

Figure 11.12 Insertion of membrane proteins with internal transmembrane sequences (Part 1)

The transmembrane α helices exit the translocon laterally and anchor proteins in the ER membrane. The polypeptides can be oriented in either direction across the membrane.

Figure 11.12 Insertion of membrane proteins with internal transmembrane sequences (Part 2)

Some proteins have an amino terminal signal sequence, and a transmembrane α helix in the middle of the protein that halts translocation and anchors the polypeptide in the membrane. The carboxy terminal portion of the growing polypeptide remains in the cytosol.

Figure Insertion of a membrane protein with a cleavable signal sequence and an internal transmembrane sequence

Proteins that span the membrane multiple times are inserted by an alternating series of internal signal sequences and transmembrane stop- transfer sequences. 47

Figure 11.14 Insertion of a protein that spans the membrane multiple times (Part 1)

Figure 11.14 Insertion of a protein that spans the membrane multiple times (Part 2)

Some proteins have a transmembrane sequence at the carboxy terminus and can’t be recognized by SRP until translation is complete. The transmembrane sequence is recognized by targeting factor TRC40, which brings the protein to a GET1- GET2 receptor in the ER membrane.

Figure 11.15 Posttranslation insertion of a protein with a C-terminal transmembrane sequence

Protein folding and processing can occur either during translocation across the ER membrane or in the ER lumen. Primary role of lumen proteins: assist folding and assembly of newly translocated polypeptides. 52

The Hsp70 chaperone BiP binds to unfolded polypeptides as they cross the ER membrane. BiP then mediates folding and assembly of multisubunit proteins.

Figure 11.16 Protein folding in the ER

Formation of disulfide bonds is important in protein folding. In the cytosol (reducing environment), most cysteine residues are in the reduced (—SH) state. In the ER, an oxidizing environment promotes disulfide (S—S) bond formation, facilitated by protein disulfide isomerase.

Proteins are glycosylated (N-linked glycosylation) in the ER while translation is still in progress. The oligosaccharide is synthesized on a lipid (dolichol) carrier. Glycosylation helps prevent protein aggregation in the ER and provides signals for subsequent sorting.

Figure 11.17 Protein glycosylation in the ER

Some proteins are attached to the plasma membrane by glycolipids called glycosylphosphatidylinositol (GPI) anchors. GPI anchors are assembled in the ER membrane and added to the carboxy terminus of some polypeptides.

Figure 11.18 Addition of GPI anchors

GPI-anchored proteins are transported as membrane components via the secretory pathway. Their orientation within the ER dictates they will be exposed on the outside of the cell.

Protein folding in the ER is slow and inefficient, and many are misfolded. They are rapidly degraded by the ER- associated degradation (ERAD) process: Misfolded proteins are identified, returned to the cytosol, and degraded by the ubiquitin-proteasome system.

Chaperones and protein processing enzymes in the ER lumen act as sensors of misfolded proteins. One pathway involves the chaperones calnexin and calreticulin, which assist glycoproteins to fold correctly.

A protein folding sensor assesses the folded glycoproteins; if correctly folded they exit the ER. If not folded correctly, the folding sensor will add back a glucose residue, allowing it to cycle back to calnexin or calreticulin for another attempt at folding.

Figure 11.19 Glycoprotein folding by calnexin

A severely misfolded glycoprotein is recognized by EDEM1, which removes mannose residues. The protein is returned to the cytosol through a ubiquitin ligase complex where it is marked by ubiquitylation and degraded in a proteasome.

If an excess of unfolded proteins accumulates, a signaling pathway called the unfolded protein response (UPR) is activated. It leads to expansion of the ER and production of more chaperones. If protein folding can’t be adjusted to a normal level, the cell undergoes programmed cell death.

Figure 11.20 Unfolded protein response (UPR)

Unfolded proteins activate 3 receptors in the ER membrane: 1. IRE1 cleaves pre-mRNA of a transcription factor (XBP1). XBP1 translocates to the nucleus and stimulates transcription of UPR genes.

2. ATF6 is cleaved to release the active ATF6 transcription factor. 3. PERK is a protein kinase that phosphorylates translation factor eIF2, which inhibits general translation and reduces the amount of protein entering the ER.

The Smooth ER Because they are hydrophobic, membrane lipids are synthesized in association with already existing membranes rather than the aqueous cytosol. Most lipids are synthesized in the smooth ER.

Eukaryotic membranes are made of 3 lipid types: phospholipids, glycolipids, and cholesterol. Phospholipids are synthesized on the cytosol side of the ER membrane from water-soluble precursors (glycerol).

Figure 11.21 Synthesis of a phospholipid

Synthesis of phospholipids on the cytosol side allows the hydrophobic fatty acid chains to remain buried in the membrane. New phospholipids are added only to the cytosolic half of the ER membrane. Some must be transferred to the other half.

This requires passage of polar head groups through the membrane, facilitated by membrane proteins called flippases. This ensures even growth of both sides of the phospholipid bilayer.

Figure 11.22 Translocation of phospholipids across the ER membrane (Part 1)

Figure 11.22 Translocation of phospholipids across the ER membrane (Part 2)

The ER is also the major site of synthesis of cholesterol and ceramide. Ceramide is converted to glycolipids or sphingomyelin in the Golgi apparatus.

Smooth ER is abundant in cells with active lipid metabolism. Steroid hormones are synthesized from cholesterol in the ER; abundant smooth ER is found in cells of the testis and ovary.

In the liver, smooth ER has enzymes that metabolize lipid-soluble compounds. These detoxifying enzymes inactivate some drugs (e.g., phenobarbital) by converting them to water-soluble compounds that can be eliminated in the urine.

Proteins and phospholipids are exported from the ER in vesicles that bud from a specialized region of the ER, the ER exit site (ERES). The vesicles fuse to form the ER–Golgi intermediate compartment (ERGIC), then move to the Golgi apparatus.

Figure 11.23 Vesicular transport from the ER to the Golgi

Proteins in the lumen of one organelle are packaged into budding transport vesicles, then released to the lumen of the recipient organelle following vesicle fusion. Membrane proteins and lipids are transported in a similar way; their topological orientation is maintained.

Proteins targeted for export have peptide and carbohydrate signals that direct their packaging into transport vesicles. Unmarked proteins in the ER can also be packaged and transported to the Golgi by a default pathway.

Proteins that function within the ER are recognized in the ERGIC or Golgi and transported back to the ER. These proteins, such as BiP, have a targeting sequence (KDEL or KKXX) at the carboxy terminus that directs retrieval back to the ER. 84

Figure 11.23 Vesicular transport from the ER to the Golgi
Repeat figure here 85

The Golgi Apparatus Golgi apparatus (Golgi complex): proteins from the ER are processed and sorted for transport to endosomes, lysosomes, the plasma membrane, or secretion. Most glycolipids and sphingomyelin are synthesized in the Golgi; and complex cell wall polysaccharides in plant cells.

The Golgi Apparatus The Golgi is composed of flattened membrane-enclosed sacs (cisternae) and associated vesicles. It has polarity: proteins from the ER enter at the cis face, usually oriented toward the nucleus. They exit from the trans face.

Figure 11.24 The Golgi apparatus (Part 1)

Figure 11.24 The Golgi apparatus (Part 2)

cis compartment—receives molecules from the ERGIC
The Golgi Apparatus The Golgi has 4 regions: cis compartment—receives molecules from the ERGIC medial and trans compartments—most modifications are done here trans-Golgi network—the sorting and distribution center

The Golgi Apparatus The mechanism of protein movement through the Golgi is an area of controversy. The stable cisternae model: proteins are carried between cisternae in transport vesicles.

The Golgi Apparatus The cisternal maturation model: proteins are carried within the cisternae, which gradually mature and progressively move through the Golgi in the cis to trans direction. Vesicles return Golgi resident proteins back to earlier Golgi compartments. 92

Figure 11.25 Transport through the Golgi (Part 1)

Figure 11.25 Transport through the Golgi (Part 2)

The Golgi Apparatus The carbohydrate portions of glycoproteins are extensively modified in the Golgi. N-linked oligosaccharides that were added in the ER are modified by a sequence of reactions catalyzed by enzymes in different compartments.

Figure 11.26 Processing of N-linked oligosaccharides in the Golgi

The Golgi Apparatus N-linked oligosaccharide of lysosomal proteins are modified by mannose phosphorylation. They are recognized by a mannose-6- phosphate receptor in the trans Golgi network, which transports them to endosomes and on to lysosomes.

Figure 11.27 Targeting of lysosomal proteins by phosphorylation of mannose residues

The Golgi Apparatus The specificity of this process is in the enzyme that catalyzes addition of N-acetylglucosamine phosphates. The enzyme recognizes a structural signal on the folded lysosomal proteins. These determinants are called signal patches, in contrast to linear targeting sequences.

The Golgi Apparatus O-linked glycosylation (carbohydrates added to side chains of serine and threonine): Processing of proteoglycans involves addition of 100 or more carbohydrate chains to a polypeptide that is further modified by addition of sulfate groups.

Figure 11.28 Structure of a Proteoglycan

The Golgi Apparatus Glycolipids and sphingomyelin are synthesized from ceramide in the Golgi. Sphingomyelin is synthesized by transfer of a phosphorylcholine group from phosphatidylcholine to ceramide. Addition of carbohydrates to ceramide yields a variety of different glycolipids.

Figure 11.29 Synthesis of sphingomyelin and glycolipids

Protein sorting and export
The Golgi Apparatus Protein sorting and export In the trans Golgi network, molecules are sorted and packaged into transport vesicles. Proteins that need to stay in the Golgi are associated with the membrane, and contain signals that prevent packaging and transport. 104

Direct transport to the plasma membrane Recycling endosomes
The Golgi Apparatus Transport from the Golgi to the cell surface can occur by three routes: Direct transport to the plasma membrane Recycling endosomes Regulated secretory pathways

Figure 11.30 Transport from the Golgi apparatus

The Golgi Apparatus Regulated pathways include release of hormones from endocrine cells and neurotransmitters from nerve cells. These proteins aggregate in the trans- Golgi network and are packaged in secretory granules. The granules store their contents until signals direct their fusion with the plasma membrane.

The Golgi Apparatus In polarized cells of epithelial tissue, plasma membranes are divided into apical domains and basolateral domains, each with specific proteins. Proteins leaving the trans Golgi network must be selectively packaged and transported to the correct domain.

Figure 11.31 Transport to the plasma membrane of polarized cells

Yeasts and plant cells lack lysosomes.
The Golgi Apparatus Yeasts and plant cells lack lysosomes. Proteins are transported from the Golgi to the vacuole, which has the same functions as a lysosome, plus nutrient storage and maintaining turgor pressure. Proteins are directed to vacuoles by short peptide sequences.

Figure A yeast vacuole

The Mechanism of Vesicular Transport
The selectivity of vesicular transport is key to maintaining the functional organization of a cell. Vesicles must recognize and fuse only with the appropriate target membrane. Understanding the mechanisms that control vesiclular transport is a major area of research in cell biology.

Three experimental approaches have been used: 1. Isolation of yeast mutants defective in protein transport and sorting 2. Reconstitution of vesicular transport in cell-free systems 3. Biochemical analysis of synaptic vesicles

1. Yeasts mutants can be defective at various stages of protein secretion (sec mutants), or are unable to transport proteins to the vacuole, or retain resident ER proteins. Isolation of mutants led to molecular cloning and analysis of corresponding genes and identification of proteins.

2. Reconstituted systems (in vitro): Enabled isolation of transport proteins, study of the transport process, and functional analysis of the proteins identified by mutations in yeasts.

3. Synaptic transmission in neurons is a specialized form of regulated secretion. Synapse: junction of a neuron with another cell. Chemical neurotransmitters are stored in the neuron in synaptic vesicles.

Stimulation of the neuron triggers fusion of synaptic vesicles with the plasma membrane and neuro-transmitters are released into the synapse. Synaptic vesicles from brain tissue can be purified in large amounts and proteins isolated.

GFP fusion proteins allow transport vesicles carrying specific proteins to be visualized by immunofluorescence as they move through the secretory pathway. Cells are transfected with cDNA encoding secretory proteins tagged with green fluorescent protein (GFP).

Figure 11.33 Visualization of transport vesicles in a living cell

Transport vesicles from the ER are coated with cytosolic coat proteins. Coats assemble as the vesicle buds off and are removed in the cytosol before it reaches its target. The vesicles fuse with the target membrane, empty their cargo, and insert their membrane proteins into the target membrane.

Figure 11.34 Formation and fusion of a transport vesicle

Three families of vesicle coat proteins: COPII-coated vesicles carry proteins from the ER to the ERGIC and on to the Golgi apparatus. COPI-coated vesicles bud from the ERGIC or Golgi and carry their cargo back, returning proteins to earlier compartments.

Clathrin-coated vesicles transport in both directions between the trans Golgi network, endosomes, lysosomes, and plasma membrane.

Figure 11.35 Transport by coated vesicles

Formation of coated vesicles is regulated by small GTP-binding proteins (ARF1 and Sar1), related to Ras and Ran. GTP-binding proteins recruit adaptor proteins that mediate vesicle assembly by interacting with cargo proteins and with coat proteins.

Figure 11.36 Formation of a clathrin-coated vesicle (Part 1)

Figure 11.36 Formation of a clathrin-coated vesicle (Part 2)

Fusion of a transport vesicle with its target: 1. The vesicle must recognize the correct target membrane. 2. Vesicle and target membrane must fuse, delivering the contents to the target organelle.

Interaction between transport vesicles and target membranes is mediated by tethering factors and small-GTP binding proteins (Rab proteins).

Table 11.1 Representative Rab Proteins

Vesicle Rab proteins in the active GTP- bound state bind membrane tethering factors. Tethering factors also bind coat proteins, and may stimulate formation of complexes between transmembrane proteins called SNAREs. 131

Figure 11.37 Vesicle docking and fusion

Figure 11.37 Vesicle docking and fusion (Part 1)

Formation of complexes between vesicle and target SNAREs is required for fusion. SNARE-SNARE pairing provides the energy to bring the two bilayers close enough to destabilize them and fuse. 136

SNARE proteins have a central coiled- coil domain. This domain binds to other coiled-coil domains and zips the SNAREs on vesicle and target membranes together. This brings the 2 membranes into direct contact and leads to fusion of the lipid bilayers.

They are the digestive system of the cell.
Lysosomes Lysosomes: membrane-enclosed organelles that contain enzymes to break down all types of biological polymers. They are the digestive system of the cell. They can vary in size and shape depending on the materials that have been taken up for digestion.

Figure 11.38 Electron micrograph of lysosomes and mitochondria in a mammalian cell

Lysosomes contain about 60 different degradative enzymes.
Mutations in genes that encode these enzymes result in lysosomal storage diseases—undegraded material accumulates in the lysosomes of affected individuals.

Lysosomes Gaucher disease is caused by deficiency of glucocerebrosidase, which catalyzes hydrolysis of glucosylceramide to glucose and ceramide. In the most common form of the disease, macrophages are the only cells affected. Their function is to eliminate aged and damaged cells by phagocytosis. 141

Molecular Medicine, Ch. 11, p. 437

Lysosomes Most lysosomal enzymes are acid hydrolases—active at pH 5 in lysosomes, but not in the cytoplasm (pH 7.2). This prevents uncontrolled digestion of cell contents if the lysosome membrane breaks down.

Lysosomes To maintain the acidic pH, a proton pump in the lysosomal membrane actively transports protons into the lysosome.

Figure 11.39 Organization of the lysosome

Lysosomes Lysosomes digest material taken up from outside the cell by endocytosis. Lysosomes are formed when transport vesicles from the trans-Golgi network fuse with a late endosome. Endosomes represent an intersection between the secretory pathway and the endocytic pathway.

Three types of endosomes: Early endosomes Recycling endosomes
Lysosomes Three types of endosomes: Early endosomes Recycling endosomes Late endosomes 147

Early endosomes fuse with endocytic vesicles from the plasma membrane.
Lysosomes Early endosomes fuse with endocytic vesicles from the plasma membrane. They separate molecules for recycling from molecules destined for degradation in lysosomes. Molecules to be recycled are passed to recycling endosomes and back to the plasma membrane.

Figure 11.40 Endocytosis and lysosome formation

Lysosomes Molecules destined for degradation are transported to multivesicular bodies and then to late endosomes. Transport vesicles carrying acid hydrolases from the trans-Golgi network then fuse with late endosomes, which mature into lysosomes.

Lysosomes Phagocytosis: specialized cells such as macrophages take up and degrade large particles—bacteria, cell debris, aged cells. Particles are taken up in phagocytic vacuoles (phagosomes), which fuse with lysosomes to become phagolysosomes.

Figure 11.41 Lysosomes in phagocytosis and autophagy

Autophagy: turnover of the cell’s own components.
Lysosomes Autophagy: turnover of the cell’s own components. Important in embryonic development and programmed cell death. A small area of cytoplasm or organelle is enclosed in a vesicle (autophagosome) which fuses with a lysosome, and its contents are digested.

Autophagy leads to continuous turnover of cellular constituents.
Lysosomes Autophagy leads to continuous turnover of cellular constituents. It can also be regulated during development and in response to stress. Example: Insect metamorphosis involves extensive tissue remodeling and degradation of cellular components. 154

Autophagy also plays an important role in programmed cell death.
Lysosomes If nutrient starvation occurs, autophagy degrades nonessential macromolecules so their components can be reutilized. Autophagy also plays an important role in programmed cell death. 155

11 Protein Sorting and Transport: The Endoplasmic Reticulum, Golgi Apparatus, and Lysosomes.

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11 Protein Sorting and Transport: The Endoplasmic Reticulum, Golgi Apparatus, and Lysosomes.

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