Chapter 4 Organelle Structure and Function

© 2020 Elsevier Inc. All rights reserved.

Chapter 4Organelle Structure and Function

© 2020 Elsevier Inc. All rights reserved. 2

Figure 4–1. Overview of plasma membrane and subcellular organelles of mammalian cell. The subcellular organelles of a typical

mammalian cell include the nucleus (surrounded by a double membrane); the rough endoplasmic reticulum (RER); the smooth

endoplasmic reticulum (SER); the Golgi complex; secretory vesicles; various endosomes; lysosomes; peroxisomes; and mitochondria

(contains an inner and an outer membrane). This example shows a simple polarized cell with apical and basolateral plasma membranes

and the tight junctions that separate them. Other junctions between cells that are not shown here are discussed in Chapter 7.


Figure 4–2. Relationship of the nuclear envelope with cellular structures. Diagram shows the double membrane that surrounds the

nuclear compartment. The inner nuclear membrane is lined by the fibrous protein meshwork of the nuclear lamina. The outer nuclear

membrane is contiguous with the membrane of the endoplasmic reticulum (ER). The outer nuclear membrane often has ribosomes

associated with it that are actively synthesizing proteins that first enter the region between the inner and outer nuclear membranes, the

perinuclear space, which is contiguous with the lumen of the ER. The double membrane of the nuclear envelope contains pores that have

regulated channels for passage of material between the cytoplasm and the nucleoplasm.


Figure 4–3. Structure of the rough and smooth endoplasmic reticulum (ER). (A) The rough endoplasmic reticulum (RER) consists of

oriented stacks of flattened cisternae studded with ribosomes on their cytoplasmic surface. The luminal space is 20–30 nm. The smooth

ER (SER) has no associated ribosomes and often appears as 30- to 60-nm-diameter membranous tubes that are connected with the

RER, so the two ER compartments share the same luminal space. (B) Electron micrograph of the RER in secretory cell from the silk gland

of the silkworm. (C) Electron micrograph of the smooth ER (SER) from a rat hepatocyte. P indicates a peroxisome and M indicates

mitochondria. ([B] From Shibata Y, Voeltz GK, Rapoport TA. Rough sheets and smooth tubules. Cell 2006;126(3):435–439. [C] Modified

from Steegmaier M, Oorschot V, Klumperman J, Scheller RH. Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth

endoplasmic reticulum membrane dynamics. Mol Biol Cell 2000;11(8):2719–2731.)


Figure 4–4. Synthesis of phospholipids in the smooth endoplasmic reticulum. Diagram presents the pathway for synthesis of

phosphatidylcholine from fatty acyl coenzyme A (CoA), glycerol-3-phosphate, and cytidine diphosphocholine (CDP-choline).


Figure 4–5. Features of signal peptides. Signal peptides typically have three functional regions. The N-terminus has a variable

length and contains positively charged amino acids at the C-terminal side. The middle region has 6–15 hydrophobic or neutral amino

acids. The C-terminal region has 5–7 residues, and positions − 1 and − 3 (where the first amino acid residue of the mature protein is + 1)

are small, uncharged amino acids that are important for proteolytic removal of the signal peptide by signal peptidase.


Figure 4–6. Testing the signal hypothesis in cell-free protein synthesis assays. (A) Messenger RNA (mRNA) for a secretory protein

is translated in a cell-free system. If microsomes (vesicles derived from the RER) are added after translation, the translated protein does

not enter the microsome, the signal peptide remains intact, and no carbohydrate is added to the protein. If a protease is added, the

protein is digested because it is not protected inside the microsomes. (B) mRNA for a secretory protein is translated in the presence of

microsomes. Ribosomes making the protein bind the microsomes, and the protein translocates to the interior of the microsome. Analysis

of the proteins indicates that the signal peptide is cleaved and the protein is N-glycosylated. If protease is added to the mixture, the

protein is not degraded because it is sheltered within the microsomes. However, if detergent is added to disrupt the membrane, the

protease and the protein come into contact, and the protein is degraded. (Adapted from Lodish H, Berk A, Matsudaira P, et al. Molecular

Cell Biology, 5th ed. New York: W. H. Freeman and Company, 2004.)


Figure 4–7. Model for translocation of secretory proteins across the endoplasmic reticulum (ER) membrane.

When the signal peptide emerges from ribosomes, the signal recognition particle (SRP) binds to the hydrophobic signal peptide (in red).

This transiently delays further elongation of the secretory protein until the SRP/ribosome complex binds to the SRP receptor on the rough

endoplasmic reticulum (RER) via interactions between the SRP and the receptor. GTP hydrolysis dissociates SRP from the complex,

which is now available to target another ribosome to the RER. Elongation of the protein resumes, and the peptide inserts into the

translocon as a loop with the N-terminus of the signal peptide on the cytoplasmic side. As elongation proceeds, signal peptidase cleaves

the signal sequence, and oligosaccharide transferase adds the preassembled N-linked oligosaccharide to Asn residues in the appropriate

context. (Adapted from Lodish H, Berk A, Matsudaira P, et al. Molecular Cell Biology, 5th ed. New York: W. H. Freeman and Company,

2004.)


Figure 4–8. Type I, II, III, and IV membrane proteins. (A) Type I proteins contain a typical N-terminal hydrophobic signal peptide that directs

ribosomes to the translocon in the rough endoplasmic reticulum (RER). Elongation of the protein results in the translocation of the N-terminus

into the lumen of the RER. When a second hydrophobic region, termed a stop-transfer anchor, enters the translocon, translocation is halted,

and the remainder of the protein is elongated on the cytosolic side of the membrane. The stop-transfer sequence causes the translocon to open

laterally releasing the protein into the bilayer resulting in an integral membrane protein anchored in the membrane by the hydrophobic stop-

transfer sequence. (B and C) Type II and III proteins do not contain an N-terminal signal sequence; rather an internal hydrophobic domain that

will eventually span the membrane serves as both the signal sequence (that binds to signal recognition particle (SRP)) and the membrane

anchor. (B) Type II proteins contain a cluster of positively charged amino acids on the N-terminal side of the anchor and insert into the

translocon as a loop, and the N-terminus remains on the cytosolic side of the membrane. (C) Type III proteins have the cluster of positively

charged amino acids on the C-terminal side of the signal anchor, which changes the orientation of the protein in the membrane. (D) Type IV

proteins have multiple membrane-spanning domains that alternate as signal-anchor and stop-transfer sequences. Two membrane-spanning

domains are shown, but the insertion mechanism can accommodate many more to produce proteins with 10 or more membrane-spanning

domains.


Figure 4–9. Hydropathy profiles can identify likely topogenic sequences in integral membrane proteins. Hydropathy profiles are

generated by plotting the total hydrophobicity of each segment of 20 contiguous amino acids along the length of a protein. Positive values

indicate relatively hydrophobic regions and negative values relatively polar regions of the protein. Probable topogenic sequences in (A)

type I, (B) type II, and (C) type IV membrane-spanning proteins are highlighted in blue. The predicted topology of multipass (type IV)

proteins, such as GLUT1 (C), must be verified using biochemical approaches. (Adapted from Lodish H, Berk A, Matsudaira P, et al.

Molecular Cell Biology, 5th ed. New York: W. H. Freeman and Company, 2004.)


Figure 4–10. Formation of a glycosylphosphatidylinositol (GPI)-anchored membrane protein. An endoprotease in the lumen of the

endoplasmic reticulum (ER) cleaves the proteins away from its C-terminal membrane-spanning domain. The new C-terminus of the

protein is attached to the amine within the ethanolamine moiety of the GPI anchor.


Figure 4–11. N-linked glycosylation in the rough endoplasmic reticulum (RER). In the early steps of N-linked glycosylation, the

oligosaccharide is preassembled on a dolichol carrier, a large, aliphatic hydrocarbon terminating in a hydroxyl group that is

phosphorylated. The oligosaccharide is assembled by one-at-a-time addition of N-acetylglucosamine, glucose, and mannose residues.

Interestingly, most of this assembly occurs on the cytosolic side of the membrane, and the oligosaccharide is then flipped to the lumenal

side of the membrane (these early steps are not shown). Once an Asn residue in the correct sequence context emerges from the

translocon, the enzyme oligosaccharide protein transferase attaches the oligosaccharide to the protein.


Figure 4–12. Calnexin and quality control in the endoplasmic reticulum (ER). As proteins emerge from the translocon, they

cotranslationally acquire N-linked sugar modifications. In addition, BiP binds and assists in the folding of the nascent protein. The N-linked

oligosaccharide of N-glycosylated proteins initially has three terminal glucose residues. Two of these glucose residues are removed by

glucosidases. Calnexin and calreticulin then bind to N-linked oligosaccharides containing a single glucose residue and in collaboration

with protein disulfide isomerase (PDI) further assist in the folding of the nascent protein. Correctly folded proteins are released, and the

last glucose residue and a mannose residue are removed, and the protein exits the ER in vesicles formed in a COPII-dependent fashion

(details described later in the chapter). However, if the protein is still misfolded, glucosyltransferase readds a single glucose residue, and

the monoglucosylated protein undergoes another round of calnexin/calreticulin-assisted folding. Proteins that cannot achieve a stable

folded conformation are shunted to the endoplasmic reticulum–associated degradation pathway (ERAD). The misfolded proteins are

delivered to the retrotranslocation channel and are exported to the cytosol. The proteins are then deglycosylated and ubiquitinated. The

ubiquitinated proteins are then targeted to the proteasome for degradation. (Adapted from Adams et al. Protein J 2019;3:317–329.)


Figure 4–13. Generic model showing the initial steps in vesicle coating, cargo recruitment, and vesicle budding. (1) The

exchange of GDP for GTP catalyzed by a guanine nucleotide exchange factor (GEF) stimulates the recruitment of a cytosolic small

guanosine triphosphate (GTP)–binding protein to the membrane. GTP-binding exposes a hydrophobic segment on the GTP-binding

protein that inserts into the membrane. (2, 3) The activated GTP-binding protein interacts with soluble coat components, recruiting them to

the membrane. There are usually two parts to the coat, the adaptor layer and the outer layer. (4) The adaptor layer binds to

transmembrane proteins that contain specific amino acid sequences that function as sorting signals. Some of the transmembrane proteins

have domains on their lumenal side that bind to soluble lumenal proteins, thus coupling the soluble proteins to sites on the membrane

where a vesicle will form. (5) The coat layers accumulate and contribute to deforming the planar bilayer into a vesicle. The vesicle

eventually buds off and the coat dissociates upon hydrolysis of the GTP bound to the small GTP-binding protein (not shown).


Figure 4–14. Vesicle tethering and fusion. (1) A coated vesicle sheds its coat and moves to the target membrane. In addition to cargo,

the vesicle contains a v-SNARE that was recruited at the time of vesicle formation. A Rab protein in the active guanosine triphosphate

(GTP)–bound form is also recruited to the cytosolic face of the vesicle. (2) The vesicle docks with the target membrane in a tethering step

that involves the Rab-GTP and a tethering protein complex on the target membrane. (3) The v-SNARE on the vesicle interacts with three

t-SNARES on the target membrane, bringing the cytosolic face of the two membranes close together so they can fuse. At some point in

this process, the Rab protein hydrolyzes GTP to guanosine diphosphate (GDP) and dissociates into the cytosol. (4) After fusion is

complete, N-ethylmaleimide sensitive factor (NSF), in association with other proteins, disrupts the v- and t-SNARE complexes freeing the

v-SNARE, which is trafficked back to its home membrane so it can participate in another round of vesicle transport.


Figure 4–15. Structure of the Golgi complex. (A) The Golgi contains stacks of flattened cisternae. These cisternae have dilated edges

from which small vesicles bud. The forming or cis face of the Golgi is the cis Golgi network (CGN), and it receives cargo from the

endoplasmic reticulum. The exporting or trans face of the Golgi is the trans Golgi network (TGN), where vesicles and cargo depart for

delivery to other membrane compartments. (B) Electron micrograph illustrating the flattened cisternae of the Golgi in a human leukocyte.

([B] Modified from https://en.wikipedia.org/wiki/File:Human_leukocyte,_showing_golgi_-_TEM.jpg.)


Figure 4–16. Overview of compartmentalized functions in the secretory pathway. Protein folding, N-linked glycosylation, and quality

control begin in the rough endoplasmic reticulum (RER). Phosphorylation of mannose on lysosomal enzymes, O-linked glycosylation, and

trimming of N-linked oligosaccharides begin in the cis Golgi network (CGN). Additional carbohydrate trimming and addition of

monosaccharides to N- and O-linked oligosaccharides occurs in the medial and trans Golgi cisternae and in the trans Golgi network

(TGN). Sulfation of proteins on tyrosine residues happens in the TGN.


Figure 4–17. Models for transport through the Golgi complex. (Left) In the vesicular transport model, cisternae are static.

Anterograde transport of cargo is mediated by COPI-dependent vesicles or tubular connections between adjacent cisternae. COPI-coated

vesicles mediate retrograde transport of cargo back to the endoplasmic reticulum. (Right) In the cisternal maturation model, (A) cargo

enters the cis Golgi, and (B–D) the entire cisterna with its resident proteins traffick to the trans face of the Golgi. Homeostasis in the

cisternal maturation model is maintained by the COPI-dependent retrograde retrieval of resident Golgi proteins, such as

glycosyltransferases, from the trans face of the organelle back to specific compartments in the Golgi where they function.

(Adapted from Martinez-Manarguez. ISRN Cell Biol 2013;2013.)


Figure 4–18. Overview of transport from the trans Golgi network (TGN). There are three main pathways for material leaving the

TGN: regulated secretion of cargo destined for the plasma membrane, constitutive secretion of cargo destined for the plasma membrane,

and the mannose 6-phosphate pathway for the delivery of lysosomal enzymes to late endosomes. Note that secretion to the plasma

membrane in polarized cells involves targeting cargo and vesicles to either the apical or basolateral domains of the plasma membrane.


Figure 4–19. Biosynthesis of mannose 6-phosphate on lysosomal enzymes. The addition of mannose 6-phosphate occurs in two

steps within the cis Golgi network. First, N-acetylglucosamine-phosphotransferase uses UDP-GlcNAc to add GlcNAc phosphate in a

phosphodiester linkage to the 6th position of mannose residues on N-linked oligosaccharides of a lysosomal enzyme. Next a

phosphoglycosidase removes the GlcNAc, leaving mannose phosphorylated on the 6 position.


Figure 4–20. Mannose 6-phosphate (M6-P) pathway for delivery of lysosomal enzymes to lysosomes. (1) Lysosomal enzymes

receive the M6-P marker in the cis Golgi network (CGN) and migrate through the Golgi cisternae with other secretory enzymes to the

trans Golgi network (TGN). (2) The M6-P receptor in the TGN binds lysosomal enzymes via recognition of the M6-P modification,

segregating lysosomal enzymes from other soluble secretory proteins. The clathrin/adaptor protein 1 (AP-1) adaptor coat is recruited by

interactions with the small guanosine triphosphate (GTP)–binding protein Arf1 (not shown) and collects M6-P receptors with associated

lysosomal enzyme cargo in the budding vesicles. The GGA adaptor is also involved in recruiting the clathrin coat to these budding

vesicles. (3) After vesicle formation the coat dissociates, and the vesicles fuse with late endosomes where the low pH dissociates

lysosomal enzymes from the M6-P receptor. (4) The lysosomal enzymes subsequently reach lysosomes when the late endosome fuses

with existing lysosomes or matures into a lysosome. (5) The M6-P receptor is returned to the TGN in vesicles that bud from the late

endosome.


Figure 4–21. Clathrin-dependent endocytosis of low-density lipoprotein (LDL). (1) The LDL receptor has an extracellular domain

that binds LDL and a cytosolic domain that binds to the adaptor protein 2 (AP-2) adaptor layer of the clathrin coat, and clathrin-coated

vesicles containing LDL receptors with bound LDL bud from sites on the plasma membrane. (2) Following internalization the coat

dissociates, and endocytic vesicles either fuse with themselves to form an early endosome or fuse with a preexisting early endosome.

The pH within the early endosome triggers the dissociation of LDL from its receptor. (3) Recycling vesicles then bud from the early

endosome (or a related structure called a recycling endosome, not shown here) carrying the unoccupied LDL receptors back to the

plasma membrane. (4) LDL in the lumen of the early endosomes goes with the fluid to the late endosome and eventually reaches the

lysosome where it is degraded, releasing its cargo of cholesterol.


Figure 4–22. Epidermal growth factor (EGF) endocytosis and the formation of multivesicular bodies. (1) EGF binds to the EGF

receptor, a transmembrane protein that is ubiquitinated on its cytosolic domain and that interacts with clathrin/adaptor protein 2 (AP-2)

adaptors. After endocytosis and dissociation of the coat, the early endosome matures into a late endosome that can also become a

multivesicular body. (2) The vesicles within multivesicular bodies are formed by invagination of the endosome membrane to form vesicles

within a vesicle that are enriched in ubiquitinated receptors, such as the EGF receptor. (3) The small vesicles within the multivesicular

body are substrates for lysosomal enzymes when the endosome fuses with lysosomes, subjecting the vesicle membrane, and eventually

the vesicle contents, to degradation. In this way, membrane-bound material originally brought into the cell by endocytic vesicles, which

includes transmembrane receptors, can be digested.


Figure 4–23. The ubiquitination pathway. (A) Free ubiquitin (Ub) is activated in an ATP-dependent reaction with the formation of a

thioester intermediate between E1 and the C-terminus of ubiquitin. Ubiquitin is then transferred to an E2, again forming a thioester

linkage. There are two classes of E3 enzymes. The RING domain E3s bind to the target (T) protein and directly transfer ubiquitin from the

E2 to the target. HECT domain E3s form a thioester intermediate between ubiquitin and itself before transferring ubiquitin to the target. An

E4 then adds a series of ubiquitins by linking through lysine 48 of one ubiquitin to the C-terminus of the next. (B) Structure of the

proteasome.


Figure 4–24 Structure of a mitochondrion. (A) Transmission electron micrograph of a mitochondrion in a human pancreatic acinar cell.

Mitochondria are surrounded by two membranes, an outer and an inner membrane. The inner membrane has many invaginations, called

cristae. Several granules can be seen in the matrix. (B) Diagrammatic presentation of mitochondrial compartments and membranes.

([A] Courtesy Keith R. Porter/Photo Researchers, Inc.)


Figure 4–25. Glycolytic pathway. The breakdown of glucose into pyruvate yields two molecules of NADH and

two molecules of ATP.


Figure 4–26. Pathway for the oxidation of fatty acids in mitochondria.


Figure 4–27. Citric acid cycle.


Figure 4–28. Summary of mitochondrial function. Pyruvate and fatty acyl coenzyme A (CoA) are transported into mitochondria by

specific transporter proteins and are metabolized to acetyl-CoA. Acetyl-CoA is then metabolized by the citric acid cycle. NADH and

FADH2 are produced by the citric acid cycle, and electrons are transferred from NADH and FADH2 to O2 by a series of electron carriers in

the inner mitochondrial membrane. A proton motive force is created by this electron transfer, and protons, moving back down their

electrochemical gradient into the matrix, power the ATP synthase to produce ATP. ATP produced in the mitochondrial matrix is

transported to the cytosol by the adenine nucleotide translocator (which exchanges ATP for ADP). Inorganic phosphate (Pi) is transported

from the cytosol into the matrix of mitochondria by the phosphate transporter.


Figure 4–29. Import of proteins into the mitochondrial matrix. Proteins are targeted to the matrix of mitochondria by amino-terminal presequences

that contain positively charged amino acids. Precursors are unfolded by cytosolic chaperones, such as heat shock protein 70 (Hsp70), before import into

mitochondria. The presequence first binds to the receptors, Tom20 and Tom22, and is then transferred to the general import pore, Tom40, of the

translocase of the outer membrane (TOM) complex. After passage through the OM, the presequence binds the intermembrane space (IMS) domain of

the Tom22 receptor protein. The presequence-containing precursor then binds the Tim50 protein and is transferred to the presequence translocase of

the inner membrane (IM; TIM23 complex). Membrane potential (ΔΨ) across the inner membrane is required for insertion of the precursor protein into

the channel formed by the Tim23 protein. The presequence translocase-associated motor (PAM), which is driven by ATP, is required for completion of

protein translocation into the matrix. The central constituent of this import motor is the mitochondrial Hsp70 chaperone (mtHsp70). mtHsp70 and its

nucleotide exchange factor, Mge1, are transiently recruited to the presequence translocase by Tim44. Pam16 and Pam18 act as cochaperones. Once

the precursor emerges in the matrix, it is bound by mtHsp70, and multiple rounds of ATP hydrolysis are required to translocate the protein across the

inner membrane. In the matrix the presequence is cleaved by the mitochondrial processing peptidase (MPP). (Adapted from Bohnert M, Pfanner N, van

der Laan M. A dynamic machinery for mitochondrial precursor proteins. FEBS Lett 2007;581:2802–2810, with permission.)


Figure 4–30. Electron micrograph of a cell showing peroxisomes. Transmission electron micrograph of a liver cell showing several

peroxisomes. A urate oxidase crystal is evident within two of the peroxisomes. (Courtesy Don W. Fawcett, Visuals Unlimited.)

Chapter 4 Organelle Structure and Function

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