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Molecular Cell Biology

Exam 3 Review Guide

Chapter 10: The Nucleus

10.1 The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm

10.1.1 Illustrate the structure of the nuclear envelope and nuclear pore complex.
 List the main functions of the nucleus. – DNA replication, transcription, posttranslational
modification, and pretranscriptional control of gene expression (regulating the entry of
transcription factors).
 The nuclear envelope is double membraned.
 The outer membrane and its properties
o The outer membrane is continuous with the rough ER
o The perinuclear space (the luminal space between the inner and outer
membrane) is directly connected with the lumen of the rough ER.
o Ribosomes are bound to the cytoplasmic side of the outer membrane.
o Rich in membrane proteins that bind the cytoskeleton.
o The outer membrane is functionally similar to the membranes of the rough ER.
o Differs from rough ER in one way: does not have proteins that give the rough ER
its tubular organization.
 The inner membrane and its properties
o It contains about 60 integral membrane proteins, such as those that bind the
nuclear lamina.
 The structure of the nuclear pore complex. List specific subunits and their functions
(120 million Daltons).
o Eight spokes around a central channel; these are connected to rings at the nuclear
and cytoplasmic sides; the spoke-ring assembly is anchored within the nuclear
envelope where the inner and outer membranes fuse.
o Cytoplasmic filaments; nuclear basket (basket-like structure);
o Central channel has FG-NUP protein (contains short subsequences of genes
(motifs)) that are rich in phenylalanine and glycine residues. (FG repeats).
 FG domains have IDRs (intrinsically disordered regions) that extend from
the central channel and form a selective barrier that controls the movement
of macromolecules.
 FG-NUPs also form the filaments that extend from the rings.
 IDRs restrict the passive diffusion of macromolecules and allow selective
passage by specialized transport proteins.
 What is the cut-off size for diffusion through the nuclear pores? 40kDa (small non-polar
molecules and protein).
 The structure of the nuclear lamina (filamentous meshwork, supports the nucleus,
made of lamins).
o Lamina
o LBR and Emerin (inner nuclear membrane); LINC complex (transmembrane)
(KASH and SUN); Lamin B receptor
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o Lamins also bind to chromatin.

10.1.2 Summarize how proteins and RNAs are transported into and out of the nucleus.
 Nuclear import
o Signals – name, make-up – Proteins that need to enter the nucleus contain nuclear
localization signals (NLS) that are detected by the nuclear transport receptors
that direct the transport through the nuclear pore complex. NLS are rich amino
acid sequences of Lysine and Arginine. Know Monopartite (continuous stretch
of amino acids) and Bipartite (two separated sections of amino acids).
o Protein that recognizes the signals- Importins
 Importins carry proteins inside the cell by recognizing NLS.
 Importins also contain hydrophobic pockets that bind the FG motifs of the
FG-NUPs with relatively low affinity, resulting in short-lived dynamic
interactions.
o Roles of Ran
 Ran is a G-protein that facilitates the movement of transport proteins from
cytoplasm to nuclear and vice versa. Ran GAP in the cytoplasm; Ran
GEF in the nucleus.
o Import process
o A protein with NLS is bound by Importin in the cytoplasm; by the hydrophobic
pocket interaction between FG motifs of the cytoplasmic filaments, importin

moves inside the nuclear through the nuclear pore complex. The hydrophobic
pockets mediate movement through the selective barrier formed by the FG motifs
of FG-NUPs that line the central channel. The Ran GTP binds to importin,
resulting in a conformational change that releases the protein inside the nucleus.
Recycling of importin Ran GTP then transports importin outside of the nucleus
into the cytoplasm, where Ran GAP hydrolyzes Ran GTP, releasing importin
back into the cytoplasm. Recycling of Ran NTF2 then binds to Ran GDP,
transporting it back to the nucleus, where Ran GEF phosphorylates Ran GDP to
recycle Ran.
 Nuclear export
o Signals – name, make-up Proteins that need to exit the nuclear contain Nuclear
Export Signals (NES) that are rich in amino acid sequences, particularly
Leucine.
o Protein that recognizes the signals Exportins
o Roles of Ran
 Unlike in importin, Ran GTP stabilizes exportin by binding to it during
nuclear export.
o Export process
o Exportins bind to cargo proteins by recognizing the NES, and Ran GTP binds to
this complex and facilitates their transport through the nuclear pore complex to
the cytoplasm. In the cytoplasm, Ran GAP hydrolyzes Ran GTP, resulting in the
delivery of cargo protein outside the nucleus. Recycling of Exportin and Ran
Finally, NTF2 carries both Ran GDP and Exportin back into the nucleus.
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 Briefly explain the export of ribosomes the 40s and 60s ribosomes are exported out of the
nucleus by exportin Crm1.
 Briefly explain the export of mRNA – mRNA is translocated from the nucleus to the
cytoplasm by the mRNA exporter complex, which binds to the mRNA after it completes
its splicing and polyadenylation. The exporter complex transports the mRNA through the
nuclear pore complex. The directionality of the process is established by RNA helicase,
which remodels how the mRNA is folded in the cytoplasmic side. Then the mRNA
exporter complex dissociates, and mRNA is released.
 Steps of transport of snRNAs between nucleus and cytoplasm, including your favorite
snurportin. snRNAs and snoRNAs are localized to the nucleus. However, upon
transcription, exportin Crm1 translocates the snRNAs to the cytoplasm, where they bind
with proteins to from snRNPs; then the importins, snurportin, translocate the snRNAs
back into the nucleus.
10.1.3 Explain how transport across the nuclear envelope can regulate gene expression.
 Briefly list two strategies.
o The transcription factor NF-κB binds to IκB in the absence of extracellular signal,
preventing its entry into the nucleus; however, under extracellular signals, the IκB
is phosphorylated and ubiquitinated by E3 ligase, and NF-κB is bound by
importin and transported inside the nucleus; therefore, this controls certain gene
expression.
o Pho4 in yeast cells helps to survive under low phosphate levels; therefore, when
there is abundant phosphate levels, Pho4 is directly phosphorylated and
deactivated. However, under low phosphate levels, Pho4 is dephosphorylated, and
this allows importin to bind and transport the transcription factor into the nucleus.
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10.2 The Organization of Chromatin

10.2.1 Explain chromosome territories the methods used to study the organization of
chromosomes within the nucleus. The nucleus is 10 μm in diameter; the DNA inside is 2m long.
 Chromatin – Carl Rabl proposed the nonrandom distribution of chromatin in the
interphase nucleus.
 Chromosomal Territories – Each chromosome was found to occupy a discrete region of
the nucleus called chromosomal territories.
 Methods of study (chromosomal territories confirmed)
o FISH (florescence in situ hybridization): uses fluorescently labelled
oligonucleotide probes that are complementary to specific repeated sequences on
individual chromosomes. This helps visualize the location of chromosome inside
the nucleus.
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o 3C (Chromosomal Conformation Capture) adjacent DNA sequence within the

same chromosome are linked and amplified to identify by high-throughput
sequencing that chromosomes occupy distinct territories inside the nucleus.
10.2.2 Summarize the relationship between transcriptional activity and chromatin localization.
 Chromosomal Organization
o Euchromatin (location): Euchromatin is decondensed and transcriptionally active
(localized in the interior of the nucleus or adjacent to the nuclear pore
complex)
o Heterochromatin (location): Heterochromatin is highly condensed and not
transcribed (associated with the nuclear membrane or the periphery of the
nucleolus)
 Constitutive heterochromatin: highly repeated sequences such as found
in centromeres and telomeres.
 Facultative heterochromatin: Not currently transcribed in the cell.
 TADs: (Topologically associated domains) looped domains.
o Each TAD domain interacts within itself, but rarely interacts with the other TADs.
o Hundred to several thousand kilobases.
o Promoters and Enhancers can interact within TAD.
o Contains regions with histone modification
o Has boundaries with CTCF and Cohesins (architectural proteins) that regulate the
organization of the genome.
 LADs: (Lamina associated domains) heterochromatin
o Chromatin immunoprecipitation technique was used to identify and isolate
regions of DNA associated with the nuclear lamins.
o 1000-1500 chromosomal domains
o 40% of human genome
o Most genes of LADs are transcriptionally repressed.
 NADs: (Nucleolus associated domains) heterochromatin
o DNA sequences found in NADs substantially overlap with those found in LADs
o Transcriptionally inactive domains are either located in LADs or NADs in
specific cells
 Heterochromatin and lamins (LADs)
o LADs bind to LBR (lamin B receptor) via heterochromatin protein 1 (HP1)
o HP1 also binds to di- or tri-methylated histone H3 lysine 9 (H3K9me2/3) residues
which are characteristic of transcriptionally inactive chromatin.
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10.2.3 Describe replication and transcription hubs.

 Replication Factories: DNA replication takes place within discrete regions called
replication factories
o Replication factories contain clusters of replication sites within which replication
of multiple DNA molecules takes place.
o BrdU staining, an analog of thymidine, can be incorporated into DNA. PCNA
(replication protein). Comparing the two-, they are present in clusters, and contain
multiple replication forks.
 Transcription Hubs are clustered sites where transcription occurs.
o Contains newly synthesized RNA, RNA polymerases, transcription factors
o Several chromosomal loci can share the same transcription hub.
o Active genes loop out and are pulled through a hub as transcription occurs.
o In some cases, co-regulated genes may be transcribed in the same hub.
o This increases the efficiency of gene expression and coordinated regulation of
related genes.
o Biomolecular condensates formed from liquid-liquid separation
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10.3 Nuclear Bodies

10.3.1 Explain the similarities and differences between nuclear bodies and cytoplasmic
organelles.
 Describe the similarities and differences.
o Similarity: Nuclear bodies compartmentalize the nucleus, concentrating specific
proteins and RNAs that function in common nuclear processes.
o Differences: Not membrane bound, but are stead biomolecular condensates
maintained by protein-protein and protein-RNA interactions. More exchange with
the rest of the nucleus
10.3.2 Describe the structure and function of nucleoli.
 Know the layered organization of the nucleolus and the streamline functions of the layers
in ribosome synthesis.
o The nucleolus is organized around the chromosomal regions that contain the
genes for the 5.8S, 18S and 28S rRNAs, which are called nucleolar organizing
regions.
o Fibrillar center (FC)
o Dense fibrillar component (DFC)
o Granular component (GC)
o Multilayer structure
 Innermost: FC
 Middle: DFC
 Outer: GC
o The genes encoding rRNA are localized in the fibrillar center and transcribed at
the interface with the dense fibrillar component where pre-rRNA is processed.
o The assembly of ribosomal subunits takes place in the granular component.
 Process:
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o Ribosomal genes are transcribed on the interface between the DFC and FC, and
the initial processing takes place in the DFC.
o Ribosomal proteins are imported into the cells to assemble on pre-rRNA; as pre-
rRNA is processed, additional ribosomal proteins and 5S rRNA are assembled to
form pre-ribosomal particles.
o The final step of processing is followed by the export of pre-ribosomal particles to
the cytoplasm, yielding the 40S and 60S ribosomal subunits.
 Briefly describe ribosome assembly.
o RNA polymerase I synthesizes a 45S ribosomal precursor RNA.
o Then it is processed to the 18S rRNA of the 40S subunit and the 5.8S and 28S
rRNA of the 60S subunit.
o 5S subunit of the 60S subunit is synthesized by RNA polymerase III outside the
nucleolus.
o Several copies of rRNA genes on 5 chromosomes

10.3.3 Compare Polycomb bodies and transcription factories.

 Know the function of polycomb bodies.
o Polycomb proteins form two multimeric complexes, PRC1 and PRC2.
o They are responsible for transcriptional repression of a variety of genes via
methylation of histone H3 lysine 27 by PRC2 and ubiquitination of histone
H2A lysine 119 by PRC1
o Polycomb proteins are concentrated in a relatively small number of regions, called
Polycomb bodies.
o Polycomb bodies are frequently associated with heterochromatin, consistent with
their role in gene repression.
o PRC1 contains intrinsically disordered regions that mediate liquid-liquid phase
separation.
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o At least some Polycomb bodies contain clusters of repressed domains from

different chromosomal regions.
 Analogous to transcription hubs
10.3.4 Summarize the functions of Cajal bodies and nuclear speckles.
 Summarize the functions.
o Both Cajal bodies and nuclear speckles are sites of snRNP assembly and storage.
o After the assembly and import, snRNPs are concentrated in the Cajal bodies,
where the final stages of snRNP maturation occur.
 Ribose methylation and pseudouridylation
o scaRNAs serve as guide RNAs to direct the ribose methylation and
pseudouridylation of snRNAs in Cajal bodies; Cajal bodies are concentration with
fibrillarin (enzymes responsible for RNA methylation).
o Role in telomerase assemblage.
o Carries out snRNA recycling to allow snRNPs to catalyze multiple consecutive
rounds of splicing.
 Be able to match the nuclear bodies with functions.

Chapter 11: Protein Sorting and Transport

11.1 The Endoplasmic Reticulum

11.1.1 Describe the mechanisms that mediate the entry and sorting of proteins into the
endoplasmic reticulum.
 Misc. details about the ER:
o ER accounts for half of all cell membranes; 10% total cell volume; RER: protein
processing and sorting; SER: lipid metabolism
 RER secretion experiment (chase):
o Pulse-chase experiment: In pancreatic acinar cells, proteins were radioactively
labelled, and in 3minute, 7minute, and 120minute intervals, the proteins were
found to be in ER, Golgi, and out of the cell, respectively.
 Secretory Pathway: RER-Golgi-Secretory-Vesicles-Cell exterior (or) RER-Golgi-Plasma
membrane or lysosome
o Free ribosomes: Protein start from cytosol to mitochondria/chloroplasts/interior of
nucleus/peroxisomes/posttranslational translocation
o Bound ribosomes: Secretion by the ribosome-ER (co-translational translocation)-
Golgi-lysosomes/plasma membrane/nuclear membrane/peroxisome membrane.
 Signal Sequences (know the “smart” experiment): Blobel finds that free ribosomes have
longer proteins compared to that in bound ribosomes inside an ER; implies that the
proteins by the bound ribosomes are cleaved. So, he hypothesizes: an amino acid signal
sequence targets the ribosome-mRNA-nascent polypeptide complex to the ER membrane,
resulting in translocation of the polypeptide into the ER lumen, whereupon the signal
sequence is cleaved from the protein by a protease.
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o Characteristic of the amino terminal signal sequence: The signal sequence is in

the N-terminus; 15-25 amino acids; 7 or more hydrophobic residues, preceded by
1-2 basic amino acids. (YOU GOT THIS!)
 SRP (signal recognition particle) and receptor
o SRP: Signal recognition particle, composed of 6 polypeptides with a small
cytoplasmic RNA (SRP RNA); has a hydrophobic pocket at its surface (used to
bind to the hydrophobic residues in the signal sequence).
o A GTPase domain interacts with the structurally similar GTPase domain of the
SRP receptor. The SRP receptor is transmembrane.
 Describe the translocon:
o Channel complex in the ER with 3 transmembrane protein called Sec61; the
largest Sec61 subunit has 10 transmembrane domains that form ring-like
structure; four of the transmembrane domains associate laterally along the length
of the translocon, forming a gate between the central channel and the
phospholipid bilayer; the cytosolic side has a ribosome binding site (through
SRP); the luminal side has a α helix that inserts into the central channel, forming a
plug, when not bound to a ribosome.
o Hairpin (reasons): the elongating polypeptide has a positively charged amino
terminus and its interaction with the negatively charged phospholipid head forms
a hairpin-like shape. The amino acid sequence draws the sequence into the
hydrophobic core of the bilayer, while the plug opens.
 Co-translational pathway using SRP into the ER lumen (DIAGRAM (refer slides if
needed 11.1 slide 16)

 Insertion of transmembrane protein into the RER membrane

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 Topology of the secretory pathway: The luminal side of the ER is the same as the
extracellular side of the cell.
 Insertion of transmembrane protein with a cleavable signal sequence (signal peptidase
cleaves it)
o The N-lumen; C-cytosolic

 Insertion of transmembrane protein without a cleavable signal sequence but with internal
transmembrane sequence
o No N-terminus signal sequence; Orientation is determined by the amino acids
immediately flanking the transmembrane domain; positively charged residues are
on the cytosolic sides because of the negative charge of the phospholipid head on
the cytosolic side.
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 Multiple transmembrane sequences

o Internal transmembrane sequences; resulting in the N terminus on the cytosolic
side.

 Post-translational insertion of tail-anchored proteins (Get and EMC) (REFERENCE

slides 11.1 23-24)
o Tail-anchored proteins: These proteins have signal sequences within 50 residues
from the C terminus; N-terminus locates in the cytosol.
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o GET pathway: Guided entry of protein by Sgt2 binding; binds to GET4-5-3

(trimeric); GET 3 ATPase transports protein to GET 1-2; GET-3 removed; protein
inserted through insertase.
o EMC: less hydrophobic polypeptide; direction recognition and insertion.

11.1.2 Explain how protein folding is facilitated in the ER and the mechanisms that are
triggered by protein misfolding.
 Protein folding in the ER lumen (Hsp70 Bip): Hsp70 chaperone Bip protein binds to the
unfolded protein that enters the ER lumen, and contributes to the folding of the protein
 Disulfide bond formation is facilitated by the enzyme protein disulfide isomerase (PDI)
in the ER lumen.

 Protein glycosylation (addition of glucose)

o Glycosylation happens on asparagine residues (N-lined glycosylation). This
process is cotranslational; an oligosaccharide unit consists of 14 sugar residues
o The oligosaccharide is transferred to the asparagine residue in Asn-X-Ser/Thr
o It is first synthesized in a lipid carrier in the ER membrane (Dolichol)
REMEMBER THIS!
o The transfer is catalyzed by oligosaccharide transferase; three glucose residues are
removed by glucosidase.
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 Addition of GPI anchors (glycosylphosphatidylinositol (GPI))

o Some proteins are attached to the plasma membrane by glycolipids; these
glycolipids are called glycosylphosphatidylinositol (GPI) anchors; GPI anchors
are assembled in the ER membrane. These are added to the C-terminus of the
protein catalyzed by an enzyme called GPI transamidase.
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 Glycoprotein Retry folding and ER-associated degradation and slow pathway

o To prevent misfolded proteins from entering the secretory pathways, ERAD (ER-
associated degradation) retries and degrades misfolded proteins.
o Calreticulin and Calnexin bind to the glycolipids when only 2 glucose resides are
removed; Calreticulin removes the third glucose and sends the correctly folded
protein to the golgi apparatus; but if the protein is misfolded, UGGT adds back a
glucose to the protein and sends it back to Calreticulin; it tries a few times; upon
repeated failure, the protein is sent to the slow pathway.
o Slow pathway: the misfolded protein is acted by slow-acting mannosidases that
remove mannose residues after prolonged misfolding; OS9 and XTP2B bind to
the misfolded protein, and interact with the retrotranslocon; the proteins are
ubiquitinized here, and degraded at the proteasome.

 UPR (three different pathways)

o UPR is activated if an excess of misfolded proteins accumulate in the ER or ER
reaches its folding capacity; results in the expansion of the ER; production of
additional chaperones; a transient reduction in the amount of newly synthesized
protein entry into the ER; or programmed cell death.
o IRE1, PERK, ATF6 (REFER SLIDES)
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11.1.3 Distinguish the roles of smooth and rough ER.

 The smooth ER (phosphatidylcholine formation): takes place in the cytosolic side; most
of the phospholipids are derived from glycerol and are synthesized from water-soluble
cytosolic precursors.
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 Translocation of phospholipids across the ER (flippase) YOU GOT THIS! Refer slides!
11.1.4 Describe transport to and retrieval from the Golgi apparatus.
 Vesicular transport from ER to the golgi
o Proteins and lipids are packaged into vesicles and transported form the ER at
ERES (ER exit site); vesicles fuse with ERGIC (ER-golgi intermediate
compartment); cargo transported to golgi; transport from cisterna-cisterna; golgi
to endosome, lysosomes, or plasma membrane;
o Resident proteins are marked with KDEL sequences (Lys-Asp-Glu-Leu) at their
C-terminus; this brings back the resident proteins back into the ER through the
retrieval pathway; Some ER transmembrane proteins are marked by short C-
terminal sequences that contain two lysine residues (KKXX).

11.2 The Golgi Apparatus

11.2.1 Relate the structure of the Golgi apparatus to its function.

 Describe the structural characteristics of the Golgi apparatus.
o The Golgi is derived from the ER, and functions to further process and sort
proteins, transport proteins and vesicles to lysosomes, plasma membrane, or
secretion, synthesize glycolipids and sphingomyelin, and synthesizes
polysaccharides of cell walls in plants.
o Polarity in structure and function: Cis face (receiving) and trans face (export).
o Compartments of the Golgi: cis, medial, trans compartment, trans-Golgi network.
 Differentiate the cisternal maturational model and the stable cisternae model.
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o Cisternal maturation model: vesicles fuse on the cis-golgi network, and the
cisternae mature and move down the golgi stack. The ERGIC then produces
another cisternae to replace that position; the compartments then mature to
become medial and trans compartments of the golgi stack. The TGN then
transports the vesicles of this matured compartment until it disappears; the
resident proteins of the golgi are then return back by transport vesicles.
o Stable cisternae model: the cisternae does not move through the stack, instead,
the proteins are carried between the cisternae by transport vesicles. The enzymes
are active within each cisternae and retained within them; or if they travel outside
the cisternae, they are retrieved by retrieval pathway.
11.2.2 Describe the types of protein glycosylation that take place in the Golgi.
 Describe the processing of N-linked oligosaccharides that are formed in the ER.
o More than 250 enzymes help in the ordered addition of sugar to the glycoprotein
as it travels through different compartments.
o REMEMBER: 14 sugar residues are added to the glycoprotein the ER (and three
glucose residues are removed; this is further modified in the Golgi; the
modification of glycoproteins depends on the glycoprotein.
 Explain how proteins are sorted to target the lysosomes.
o Proteins destined for incorporation into lysosomes are modified by mannose
phosphorylation.
o In cis compartment, N-acetylglucosamine phosphates are added to specific
mannose residues.
o Then the N-acetylglucosamine group is removed, leaving mannose-6-phosphate
residues on the N-linked oligosaccharide.
o In the trans-Golgi network, M6P is recognized by mannose-6-phosphate
receptor, which directs the transport of these proteins to endosomes and on to
lysosomes.
 Describe the process of O-linked glycosylation in the Golgi apparatus. (THE LINK is on
Oxygen)
o Proteins can be modified by the addition of carbohydrates to the sidechains of
acceptor serine/threonine residues within specific amino acids. This process is
called O-linked glycosylation. Some O-links consist of only few sugar residues,
while others are long chains of sugar. This is added in the Golgi.
 Know that proteoglycans are formed using O-linked glycosylation.
o Proteoglycans contain long chains of glycosaminoglycans (GAGs), which are
repeating disaccharides (e.g., chondroitin sulfate and keratan sulfate), joined to a
core protein. Each GAG many contain up to 100 sugar residues and as many as
100 GAGs may be linked to a core protein.
11.2.3 Summarize the role of the Golgi in synthesis of membrane lipids.
 Know that ceramide is synthesized in the ER and is the precursor of sphingolipids and
glycolipids.
 Know how sphingolipids and glycolipids are formed in the Golgi.
o Sphingolipids: Sphingomyelin is synthesized by the transfer of a
phosphorylcholine group from phosphatidylcholine to ceramide.
o Sphingomyelin is synthesized on lumenal surface of the Golgi.
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o Glucose is added to ceramide on the cytosolic side.

o Glucosylceramide flips to the lumenal side.
o Carbohydrates are added to the lumenal side.
o Eventually glycolipids are on the exterior of the plasma membrane
o SIMILARLY, adding different carbohydrates to ceramides can yield a
variety of different glycolipids.
11.2.4 Diagram the routes of protein export from the Golgi.
 Know how proteins are sorted in the trans-Golgi network to different destinations,
including different microdomains of the plasma membrane.
o Pathway to lysosomes: YOU KNOW IT, HANDEL; M6P
o Secretion and transmembrane proteins that function at the plasma membrane:
 Route 1: direct transport from the trans-Golgi network to the plasma
membrane, continuous secretion or incorporation.
 Route 2: via recycling endosomes
 Route 3: regulated secretory pathway – specific proteins are secreted in
response to environmental signals.
 Immature secretory granule.
 Proteins aggregate in the TGN, pH about 6.4
 Further condensed in the immature secretory granules, which
develops into mature secretory granules.
 Signal directs release.
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 Transport to the plasma membrane of polarized cells

o Epithelial cells contain apical and basolateral domains.
o Distinct domains of the plasma membrane are present in other cell types too.
o Selective packaging: specific proteins need to be packed specifically for their
function in the distinct membranes.
o Basolateral domain: dileucine (LL) or tyrosine-containing hydrophobic motifs,
within their cytoplasmic domains.
o Apical domains: GPI anchors and carbohydrate modifications.

11.3 The Mechanism of Vesicular Transport

11.3.1 Summarize the process of vesicular transport.

 Know the different routes of vesicular trafficking (IMAGE)

 Overview of vesicular transport
o Budding - A vesicle bud forms from the membrane of one compartment (the donor
membrane).
o Packaging - The packaging of specific soluble and transmembrane proteins into the
growing bud.
o Pinching off - As the bud grows, it ultimately pinches off the donor membrane, yielding
a transport vesicle.
o Trafficking - It is transported along cytoskeletal filaments to a target membrane.
o Fusing - The vesicle fuses with the target membrane, whereupon the transmembrane
proteins are incorporated into that membrane and the soluble proteins are released into
the lumen of the target compartment of the extracellular space.
11.3.2 Explain how vesicle coat proteins contribute to vesicle budding.
 Transport by coated vesicles
o COPII: From ER to ERGIC to Golgi apparatus; transitional ER forward.
o COPI: From ERGIC or Golgi back to the earlier compartments; retrieval.
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o Clathrin: In both directions between TGN, endosomes, lysosomes, and the plasma
membrane.

 Arf 1 and Clathrin coated vesicles

o Clathrin: Clathrin is made of triskelions with three flexible arms that extend from the
central hub at an angle.
o Triskelions form an interlaced complex on the surface of a membrane, forming a curved
lattice structure with its concave face lying against the membrane.
o Arf 1: Arf 1 is a small G-protein that is anchored on the cytosolic side by a myristoyl
group.

11.3.3 Link the processes of vesicle budding and the recruitment of cargo proteins to the production of
transport vesicles
 Formation of Clathrin coated vesicles at Trans Golgi Network (TGN)
o Arf1/GDP becomes Arf1/GTP by Arf GEF.
o Arf1/GTP recruits an adaptor protein to the membrane.
o The adaptor protein binds to the sequences in the cytosolic domains of the cargo receptor.
o The receptor selectively binds specific cargo in the lumen.
o The adaptor protein also binds clathrin triskelion, initiating assembly of the vesicle coat.
o Formation of clathrin-coated vesicle.
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o Dynamin pinches off the vesicle.

 Dynamin:
o Dynamin is large G protein that assembles into helical polymers around the neck of
budding vesicles.
o It couples GTP hydrolysis to constrict, in turn driving membrane fission.
11.3.3 Describe the mechanism by which vesicles dock and fuse with the correct target membranes.

 Vesicle docking and fusion

o Docking
 Rab – a small G-protein- is attached to the transport vesicles and attaches to the
Rab tethering factor at the target site. There are more than 60 Rab proteins that
specify different organelles and transport vesicles.
o Fusion:
 When a v-SNARE interacts with a t-SNARE, the helical domains of one wraps
around the helical domains of the other.
 They form a very stable four-helix bundle, the trans-SNARE complex.
 The trans-SNARE complex locks the two membranes together.
 Complete process in order:
o Tethering: Rab proteins on the vesicle membrane binds to the tethering factor at the target
membrane.
o Docking: The formation of complexes between SNAREs on the vesicles and target
membranes.
o The protein coat is removed; the coiled-coil domains of the SNARES zip together, bring
the vesicles and the target membranes to close proximity.
o The membranes fuse.
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 Know SNARE complexes in docking

o V-SNARES: vesicular SNARES (found on vesicles) single polypeptide chain.
o T-SNARES: target SNARES (found on target membranes) three proteins.
o THE V and T- SNARE binding is very specific; this makes sure that the vesicles bind to
the correct target membranes.
11.4 Lysosomes

11.4.1 Describe the function of lysosomes.

 Misc. Information about lysosomes
o Membrane-enclosed organelles that contain enzymes capable of breaking down
biological polymers (proteins, nucleic acids, carbohydrates, and lipids); digests stuff from
outside the cell and cellular components.
 Morphology of Lysosomes
o Morphologically diverse organelles; their shape is determined by what they are
degrading.
 Acid Hydrolases
o Lysosomes contain 60 degradative enzymes that hydrolyze proteins, DNA, RNA,
polysaccharides, and lipids.
 Discuss about the diseases caused from malfunction of lysosomes: Due to lack of enzymes (due to
mutations in genes), undegraded material can accumulate within the lysosomes; causing over 30
different diseases. Examples:
o Gaucher disease: mutation in genes that encode for enzymes required for the breakdown
of glycolipids; deficiency in glucocerebrosidase; enzyme replacement therapy:
exogenous enzymes are taken up by macrophages with receptors that recognize mannose
residues.
o I-Cell disease: A deficiency in the enzyme that catalyzes the first step in the tagging of
lysosomal enzymes with mannose-6-phosphate in the Golgi apparatus. The result is a
general failure of lysosomal enzymes to be incorporated into lysosomes.
 Organization of lysosomes: Most lysosomal enzymes are hydrolases
o These enzymes are active at the acidic pH 5
o The cytosolic pH is 7.2; so, this protects cellular components from accidental digestion if
lysosome leaks.
o Protons are transported into the lysosome by a proton pump, and this requires ATP
hydrolysis.
11.4.2 Explain how lysosomes are formed.
 Lysosomes formation and endocytosis
o Lysosomes digest material taken from the outside of the cell (endocytosis); the vesicles
from the golgi fuse with late endosomes, forming the lysosome.
 Secretory pathway – lysosomal proteins are processed
 Endocytic pathway – extracellular molecules are taken up at the cell surface.
o Vesicles formed by endocytosis at the plasma membrane fuse with early endosomes,
which separate molecules targeted for recycling back to the plasma membrane from those
destined for degradation in lysosomes.
o The molecules to be recycled are then passed to recycling endosomes and back to the
plasma membrane.
o Molecules destined for degradation are transported to late endosomes, which develop into
lysosomes as they receive lysosomal enzymes from the Golgi apparatus.
o The material taken up by endocytosis is then degraded.
11.4.3 Summarize the process of autophagy.
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 Autophagy: degradation of own cell’s components (digests proteins with cellular components such
as mitochondria).
o Autophagosome: double membrane structure which fuses with the lysosome
o Autolysosome: the final product after the fusion of autophagosome and lysosome within
which the encapsulated materials are digested.

 ATG and autophagosome formation

o Production of vesicles from the TGN or endosomes that are packaged with the
transmembrane protein Atg9.
o These vesicles dock at the ER membrane via interactions between the Atg9 cytosolic
domain and a scaffolding complex on the ER composed of several Atg proteins.
o The Atg9 vesicles fuse, forming an autophagosome precursor.
o The autophagosome precursor expands by two routes:
 Direct transfer of lipids to the autophagosome from the ER membrane, catalyzed
by the lipid transfer protein Atg2.
 Fusion with vesicles derived from other sites on the ER.
o The autophagosome precursor membrane expands. It adopts a concave shape called a
phagophore.
o The phagophore continues to expand and grows into a double-membrane spherical
structure within which cytoplasmic substances become encapsulated.
o The spherical phagophore ultimately closes, yielding an autophagosome that is released
from the ER membrane before it fuses with a lysosome.

 Other Atg proteins

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o Eight Atg proteins work together to carry out a sequence of chemical reactions that
culminate in the covalent linkage of Atg8 to phosphatidylethanolamine (PE) within the
membranes of phagophores.
o Atg8-PE appears to play multiple roles during membrane expansion, including the
binding and recruitment of specific substances into phagophores that are marked with
tags for autophagic degradation.
 Misc. information about autophagy
o Autophagy maintain cells’ homeostasis; as cytoplasmic proteins incur damage, the slow
and non-selective degradation by lysosomes removed such damaged stuff.
o 1% of cell proteins per hour are degraded by this mechanism, balanced by the synthesis
of new ones.
 Mitophagy (degradation of mitochondria)
o Mitophagy is prompted by the linkage of phosphorylated ubiquitin tags to proteins on
their outer membrane.
o These tags are recognized and bound by proteins termed autophagy receptors that also
bind Atg8-PE, which in turn recruits the damaged mitochondria into expanding
phagophores for degradation.
 Autophagy providing cells with nutrients
o Cells also couple autophagy levels to the availability of ATP and other nutrients (e.g.,
amino acids and glucose).
o If ATP or other essential nutrients drop too low, autophagy is up-regulated to replenish
the supply by degrading macromolecules and recycle the components.
 Parkinson’s and lysosomes
o The mutation in PINK1 and parkin genes, which encode for the tagging of mitochondria
with ubiquitin for degradation, can cause Parkinson’s disease.

Chapter 12: Mitochondria, Chloroplasts, and Peroxisome

12.1 Mitochondrial Structure, Composition, and Dynamics

12.1.1 Illustrate the functional organization of mitochondria.

 Know the structures.
o
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12.1.2 Compare and contrast the characteristics of the two mitochondrial membranes and how
their characteristics relate to their roles.
 Know the different extents of permeability.
o Inner Membrane: Impermeable to most ions and small molecules; contains most
of the enzymes for oxidative phosphorylation and metabolites for those reaction
in the matrix. Has cardiolipins. True protective barrier.
o Outer Membrane: Highly Permeable; has porins. The composition of the
intermembrane space is similar to the cytosol with respect to ions and small
molecules; its integrity plays a critical role in regulating apoptosis (cytochrome
c); molecules <1000kDa can passively diffuse through porins
 Know that the cardiolipin is located in the inner membrane.
o Matrix: it contains the mitochondrial DNA and the ribosomes; very high protein
concentration in the matrix, at more than 500 mg/mL, close to that in a protein
crystal; DNA is organized into compact bodies - the nucleoids - by special
scaffolding proteins that also function as transcription regulatory proteins.
12.1.3 Describe the dynamic nature of mitochondrial networks resulting from mitochondrial
fusion and fission.
 Describe the process of mitochondrial fusion.
o Mitochondrial fusion involves Mfn1 and Opa1; the Mfn1 and Opa1 are GTPase
proteins.
o Mfn1 is found on the outer membrane of the mitochondria; the Mfn1 of two
mitochondrion due to the protein-protein interaction, along with the GTPase
activity hydrolyzes GTP to GDP. Conformational change pulls the two together,
resulting in the fusion of the outer membrane.
o Opa1 plays the key role in fusion of the inner membrane; the Opa1 of the inner
membrane binds with the cardiolipins of the other mitochondria (and vice versa);
hydrolysis of GTP to GDP; the inner membranes fuse.

 Describe the process of mitochondrial fission.

o Mitochondrial fission involves Drp1, another GTPase protein, recruited by the
transmembrane adapter proteins;
o It forms a ring structure around the mitochondrion; hydrolysis of GTP to GDP;
contractile ring pinches the mitochondria, leading to mitochondrial fission.
 Explain how fusion and fission mains the mitochondrial networks.
o In most cells, mitochondria exist as networks that vary in organization based on
cell type and other conditions.
 Continuous fusion and fission remodels the network.
 Fusion allows exchange of genetic materials.
 Fission is important in distribution to daughter cells.
 Fission facilitates the transport of mitochondria to areas of the cell where
production of energy is in high demands.
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12.2 Oxidative Catabolism of Glucose and Fatty Acids

 This part is not much more than cell biology. Just use the learning objectives to review
the process.
 You don’t need to memorize the structures and names of all the intermediates. Just know
the key reactions.
12.2.1 Summarize the reactions through which glucose is catabolized during glycolysis and the
citric acid cycle and how they contribute to ATP production.
 Stage 1. Glycolysis: common to all cells; independent of oxygen; in the cytosol; output:
2 Pyruvate, Net gain of 2 ATP, and 2 NADH.
 Early reactions of glycolysis consume 2 ATP; 4 ATP produced, so net gain is 2 ATP.
Reduction of NAD+ to NADH.
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 Oxidative carboxylation of pyruvate: Pyruvate is oxidized in the matrix by the Coenzyme

A; output: Acetyl CoA, NADH, CO2, and No ATP. The Acetyl CoA then enters the
citric acid cycle; output: 4 molecule of ATP, 10 molecules of NADH and 2 molecules of
FADH2; reoxidation of NADH and FADH2 yields ATP by transferring an electron
through ETC to reduce to O2 and H2O. Each NADH molecule yields 3 ATP, and each
FADH2 yields 2 molecules of ATP. Total yield is 38 molecules of ATP.
 OUTPUTS: 4ATP from glycolysis, 30 from NADH and 4 from FADH2.
12.2.2 Describe the breakdown of fatty acids and how they contribute to ATP production.
 Fatty acids produce WAY more ATP than glucose. They undergo cyclic oxidative
process; Each oxidation event yields 1 molecule of NADH, 1 molecule of FADH2, and a
molecule of acetyl CoA and fatty acyl CoA.
 Net gain is of a 16-carbon fatty acid is 7 molecules of NADH, 7 molecules of FADH2,
and 8 acetyl CoA.

12.2.3 Compare the relative yields in ATP production between glucose and fatty acid
catabolism.
 16-carbon fatty acids
 7 NADH (3X7=21 ATP)
 7 FADH2 (2X7=14 ATP)
 8 acetyl CoA (12X8=96 ATP)
 One ATP was used to start the process.
 Gain: 130 ATP
 6-carbon glucose – 38 ATP
 16C saturated fatty acid: 256 g
 6C glucose: 180g
 There is 2.5 times more ATP per gram by fatty acids.
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12.3 Oxidative Phosphorylation

 Just use the learning objectives.

12.3.1 Compare the mechanisms of ATP formation during glycolysis and oxidative
phosphorylation.
 The transfer of electrons in the ETC helps to create a proton gradient across the inner
membrane. This helps the proton move down the gradient through ATP synthase, which
yield ATP as a result from ADP and inorganic phosphate. (DURING Oxidative
Phosphylation).
 These two stages occur parallel within the mitochondria, ensuring a continuous supply of
ATP in the cell.
 Substrate-level phosphorylation: A high-energy phosphate is transferred from a substrate
of an energy-yielding reaction directly to ADP. (DURING Glycolysis).
12.3.2 Explain chemiosmotic coupling.
 Two way of ATP production:
o Chemiosmotic coupling: The potential energy stored in the proton gradient is
harvested by ATP synthase. (DURING oxidative phosphorylation).
12.3.3 Describe how the transfer of electrons from NADH and FADH2 to O2 yields an
electrochemical gradient.
 Know the components of the electron transport chain.
o Electron from NADH transport:
 Complex I (NADH dehydrogenase) acts as a proton pump; electrons
transferred from NADH to Flavin mononucleotide through iron-sulfur
carriers. Four protons are pumped out here.
 Coenzyme Q (ubiquinone) is reduced to QH, and carriers electrons and 4
H+ to Complex III.
 Complex III transfers the electrons to cytochrome c, and pumps 4
electrons into the intermembrane space.
 Cytochrome c transfers the electrons to Complex IV, which transfers them
to O2 to form H2O, pumping two more protons out of the matrix.
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o Electrons from FADH2 transport

 Electrons form FADH2 enter the ETC at complex II (succinate reductase),
and are then transferred to coenzyme Q; no proton is pumped in this step.
 Protons are pumped only in complexes III and IV because free energy is
released, exactly like NADH transport process.

12.3.4 Illustrate how a proton gradient across the inner mitochondrial membrane is used to
produce ATP.
 The proton gradient created so far created an electric voltage difference across the
membranes (inner membrane and matrix); moreover, the presence of a proton creates a
chemical gradient due to the change in pH across the matrix and the intermembrane space
creating an electrochemical gradient.
 The matrix is more negative than the intermembrane space; the pH of the intermembrane
space is higher by 10 times than the pH in the matrix.
 This electrochemical gradient is used by Complex V (ATP synthase) to produce ATP.
 F0 is an electrically driven motor that spans the membrane and provides a channel
through which protons are able to flow down their gradient into the matrix.
 F0 and F1 are linked by a peripheral stalk.
 Central stalk of F1 spinning to synthesize ATP, as a rotary motor; F0F1ATP
 4 protons for 1 ATP
 1 NADH for 3 ATP
 1 FADH2 for 2 ATP

12.4 Mitochondrial Assembly and Maintenance

12.4.1 Describe mitochondrial genomes and their contribution to the protein composition of
mitochondria.
 Know the categories of proteins encoded by the mitochondrial genome.
o A small number of proteins (13) essential for mitochondrial oxidative
phosphorylation is encoded by mitochondrial genome.
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o rRNAs and tRNAs (16S and 12S rRNA; 22 tRNAs) needed for translation of their
protein-coding sequences within mitochondria are also encoded by mitochondrial
genomes.
o Nuclear genes encode mitochondrial RNA polymerase, ribosomal proteins, and
translational factors.
 Know how the mitochondrial genome uses genetic codes differently.
o Mitochondria uses a slightly different genetic code than the universal code; the
wobble rule (multiple codons can code for a single amino acid) requires at least
30 different tRNAs, but the mitochondria has only 22 tRNAs, showing that the
“U” in the anticodon can bind to all the four codons recognized by a single tRNA.
o Some codons specify different amino acids in mitochondria than in the universal
code.
 Know mitochondria replacement therapy
12.4.2 Summarize how proteins and lipids are imported into mitochondria.
 Background: Since 99% of the mitochondrial proteins are encoded in the nucleus and
made by cytosolic free ribosomes and transported to the mitochondria after translation
(posttranslational translocation),there are multiple way multiple proteins get imported
into the mitochondria.
 Describe the presequence.
o The N-terminal presequences of 15-55 amino acids direct the import of proteins
to the matrix and inner membrane.
o They contain multiple positively charged amino acid residues, usually in α helix,
and are usually removed by proteolytic cleavage following their import into the
organelle.
 Know the process of presequence-dependent import to the matrix and inner membrane.
o Presequence binds to the Tom complex; kept unfolded by the chaperone protein
Hsp70 in the cytosol. Using ATP hydrolysis, the protein translocates across the
outer membrane; proteins are then transferred to Tim23 complex.
o If proteins contain transmembrane sequences, they go laterally into the inner
membrane and exit the Tim23 complex.
o Hsp70 in the matrix of the mitochondria is part of an import motor complex that
drives protein import; the presequence is then cleaved by MPP (matrix processing
peptidase), and the protein binds to other chaperone proteins.
o An electrochemical gradient is necessary for transport into the matrix; this is
achieved by the ETC. The matrix is negative, and the presequence carries
positive charges.
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 Know how multipass transmembrane proteins are targeted to inner membrane by

district signals.
o Proteins without presequence have distinct signal sequences internally that help
them travel through the Tom complex; however, instead of the Tim23 complex,
Tim9-10 proteins attach to the protein in the intermembrane space and escort the
protein to the Tim22 complex.
o If it contains a transmembrane sequence, the protein exits Tim22 and is embedded
in the inner membrane.
o Proteins encoded by the mitochondrial ribosomes are translocated to the inner
membrane by Oxa1 after being carried by chaperone proteins (Hsp70).
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 Sorting of proteins to the outer membrane and intermembrane space

o Signal transmembrane domain proteins destined for the outer membrane are
inserted on the outer membrane via the Mim1 complex.
o Pre-cursors of the β-barrel complex enter through the Tom complex, and the Tim9-10
chaperones carry the protein to the SAM (sorting and assembly machinery) complex,
where they are inserted on the outer membrane.
o Proteins destined for the intermembrane space are translocated via the Tom
complex, but are retained in the intermembrane space by specific intermembrane
chaperone proteins.
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 Describe the process of phospholipid transfer from the ER to the mitochondria.

o The phospholipid transfer is done in regions where the ER and mitochondria are
in close contact; phospholipid transfer protein carry single phospholipid
molecules from the ER to the mitochondria.
o In animals, sphingolipids, cholesterol, phosphatidylcholine, phosphatidylinositol
and phosphatidylserine are synthesized in the ER and transported to mitochondria.
o Mitochondria catalyze the synthesis of cardiolipin, which is found only in the
inner membrane.
o The mitochondria synthesize phosphatidylethanolamine from phosphatidylserine.
12.4.3 Explain the role of the proton gradient in the transport of proteins and metabolites
across the mitochondrial membrane.
 Explain protein transport driven by proton gradient.
o Requires the proton gradient
o Transfer of ATP and ADP across the inner membrane is mediated by adenine
nucleotide translocator (ANT)
o 1 ADP into the matrix in exchanged for 1 ATP out of the matrix
o Driven by the electric gradient
o ATP carries -4, ADP -3
o ATP is more negative, going out, so the process is driven by the voltage
component of the mitochondria.
 Explain metabolite transport driven by protein gradient.
o Transfer of Pi for synthesis of ATP mediated by another transport protein
o Import phosphate (H2PO4-) and exports hydroxyl ions (OH-)
o Transport is driven by the concentration gradient
o OH- going to the cytosolic side and Pi is moving into the matrix.
o The import of pyruvate from the cytosol is mediated by a transport protein that
exchanges pyruvate for hydroxyl ions.

12.5 Chloroplasts and Other Plastids

12.5.1 Compare the structural and functional organization of chloroplasts with mitochondria as
well as their respective genomes.
 Know the structural characteristics of chloroplasts.
o 5-20 μm long; chloroplast envelope is double membrane, but there is a third
internal membrane called the thylakoid membrane.
o The thylakoid membrane forms thylakoids; stacks of thylakoids are called grana.
o There are three compartments within the membranes: intermembrane space,
stroma, and thylakoid lumen.
o The outer membrane of the chloroplast is permeable to small molecules and it
contains porins. The inner membrane is impermeable to small molecules and
metabolites and requires membrane transporters.
o The stroma is like the mitochondrial matrix: It contains the chloroplast genetic
system and a variety of metabolic enzymes, including those responsible for the
dark reactions.
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o There are multiple photosystems and NADP reductase.

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 Compare with mitochondria.

o OBSERVE THE GRADIENTS (lumen is more concentrated than the
stroma; similarly, the stroma=matrix; lumen=intermembrane space.
12.5.2 Explain how energy from sunlight is harvested by chlorophyll and used to drive
production of ATP and NADPH via the light reactions in chloroplasts.
 Describe the electron transport chain from PS II to NADP reductase.

 Know the linear and cyclic electron flow.

o Linear flow: regular
o Cyclic flow: PSI and Cytochrome bf loop (production of 1ATP, but no NADPH;
less production of ATP than linear flow.
 Know the resonance energy transfer.
o Chlorophylls are organized into photocenters, which contain hundreds of
chlorophylls that act as antennae to absorb the light energy and convert it into
chemical energy.
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o The absorption of photons by a chlorophyll molecule excites an electron within its

porphyrin ring to a higher energy state.
o This converts light energy into potential chemical energy.
o The electron returns to the normal state.
o An adjacent chlorophyll absorbs the released energy.
o One to the next.
o Eventually the energy is transferred to a pair of chlorophyll molecules in the
reaction center of photosystem II.
 Know the process of ATP production.
o YOU GOT THIS!
 Know the difference between the proton gradients across the mitochondrial inner
membrane and the thylakoid membrane.
o The thylakoid membrane though impermeable to protons, is permeable to Mg2+
and Cl- ions; this neutralizes the electric gradient across the membranes, leaving
with only the chemical nature across the membranes. Therefore, unlike the
mitochondria which has an electrochemical gradient, the thylakoid lumen and the
stroma have a chemical gradient; however, this different is greater; more than 3
pH difference.
o For each pair of electrons transported, 2 protons are introduced into the thylakoid
by splitting water, and 4 are pumped by cytochrome bf.
o 4 protons are required to produce 1 ATP
o Linear electron flow yields 1.5 ATP
o Cyclic flow yields 1 ATP
12.5.3 Summarize how the Calvin cycle reactions produce glucose.
 Do not worry about the details of the intermediates. Just the major inputs and outputs.
o The dark reactions take place in the stroma.
o NADHP and ATP are used to synthesize glucose from CO2 and H2O.
o The Calvin cycle consumes 18 ATP and 12 NADPH.
o It requires 24 electrons pass through the ETC.
o Electrons from splitting 12 H2O to form 6 O2
12.5.4 Summarize the mechanisms of protein import into chloroplasts.
 Describe the process of protein import into the stroma in steps.
o Hsp70 keeps the protein in an unfolded state.
o Some Toc proteins bind and hydrolyze GTP, providing an additional source of
energy for translocation.
o After the protein gets into the outer membrane, it is transferred to the translocon
of the inner membrane, the Tic complex into the stroma.
o A transmembrane of Tic links to Toc, keeping the two complexes associated at
sites of close contact.
o In the stroma, Hsp93 chaperone is associated with the stromal side of the Tix
complex.
o Hsp93 draws the protein through the inner membrane.
o In the stroma, the transit peptide is cleaved by a stromal processing peptidase
(SPP).
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o Then the protein associates with stromal Hsp70 chaperones.

o Transmembrane protein of the outer membrane exits Toc laterally.
o Inner membrane protein
o exits Tic laterally
o gets into the stroma and transfers through another translocon in the inner
membrane
o Intermembrane space protein is transported into Toc but do not translocate into
the Tic complex.

 Explain the roles of different parts.

o Transit peptides: N-terminal 30-100 amino acid sequences that act as signal
sequence for translocation into the stroma.
o Toc complex: Outer membrane
o Tic complex: Inner membrane
o Hsp93: In the stroma, Hsp93 chaperone is associated with the stromal side of the
Tic complex. Hsp93 draws the protein through the inner membrane.
o SPP: Stromal processing peptidase that cleaves the transit peptides.
 Describe the three pathways by which proteins are imported into the thylakoid lumen
from the Stroma.
o TAT pathway (twin arginine translocation):
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Protein is bound by proteins STT1 and 2 in the stroma, which recognize a

twin-arginine signal sequence within substrate and promote liquid-liquid
phase separation via their intrinsically disordered regions.
 It appears that the resulting condensate facilitates delivery of substrates to
the multi-subunit Tat complex.
 Interactions between STT1/2 with Tat subunits destabilize the condensate.
 Release and transport through Tat
 Proteolytic cleavage by thylakoid processing protease (TPP).
o Sec Pathway:
 Signal sequence is recognized by the SecA protein
 Translocate into the Sec translocon
 Transmembrane protein can exit Sec translocon laterally
o SRP Pathway:
 Proteins are incorporated into the thylakoid membrane via the Alb3
insertion machinery.
 Related to Oxa1 in mitochondrial inner membrane
 Signal sequence is recognized by a chloroplast signal recognition particle
(cpSRP).
 The cpSRP delivers the protein to Alb3.
 The cpSRP can also recognized proteins produced in the stroma.

 Know the roles of different parts.

o STT1 and 2: form complex to carry protein to Tat pathway; dissociate upon
contact with Tat complex.
o TPP: cleaves the signal sequence.
o SecA: protein that facilitates the translocation of protein in the Sec pathway
o cpSRP: chloroplast signal recognition particle that binds to the signal sequence of
the protein in the SRP pathway.
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o Alb3: Similar to Oxa1; translocon of the SRP pathway.

12.5.5 Describe the roles of other plastids.
 Names and roles.
 Origin.
 Interchangeability. REFER SLIDES

12.6 Peroxisomes

12.6.1 Summarize the roles of peroxisomes in animal and plant cells.

 List the functions in animals and plants.
o In animal cells, fatty acids are oxidized in both peroxisomes and mitochondria.
o However, in yeasts and plants, fatty acid oxidation is restricted to peroxisomes.
o Plants: Peroxisomes in seeds are responsible for the conversion of stored fatty
acids to carbohydrates, which is critical to providing energy and raw materials for
growth of the germinating plant.
o Peroxisomes in leaves function coordinately with chloroplasts to metabolize a
side product of Calvin cycle, through which CO2 is converted to carbohydrates
during photosynthesis.
o Animals: Biosynthesis of lipids
o In animal cells, cholesterol and dolichol are synthesized in both peroxisomes and
ER.
o In the liver, peroxisomes are involved in the synthesis of bile acids, which are
derived from cholesterol.
o Especially enzymes required for synthesis of plasmalogens
o A family of phospholipids in which one of the hydrocarbon chains is joined to
glycerol by an ether bond rather than by an ester bond.
o Important membrane components in some tissues, particularly heart and brain,
although they are absent in others.
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 Describe how the peroxisomes oxidize fatty acids and decompose hydrogen peroxide.
o Peroxisomes oxidize fatty acids and decompose hydrogen peroxide using
catalase.
o The oxidation of a fatty acid is accompanied by the production of hydrogen
peroxide (H2O2) from oxygen. The hydrogen peroxide is decomposed by catalase,
either by conversion to water and oxygen or by oxidation of another organic
compound (represented as AH2).
12.6.2 Describe the pathways responsible for peroxisome biogenesis.
 Import of the transmembrane protein from the ER.
o Transmembrane proteins are imported from the ER.
o Metabolites
o Peroxins or Pex proteins are involved in peroxisome assembly.
o Peroxisomal transmembrane proteins are derived from the peroxisomal ER. Two
different types of vesicles (designated V1 and V2) carrying distinct transmembrane
proteins fuse to form a functional peroxisome. The combination of these different
proteins forms the machinery (importomer) through which internal peroxisome
proteins synthesized on cytosolic ribosomes can be imported.
 Import of the matrix protein from the cytosol.
o Peroxisomal matrix proteins containing the targeting signal PTS1 are recognized
by the cytosolic receptor Pex5. The Pex5/cargo complex then binds to a docking
complex (Pex13, Pex14, and Pex17) on the peroxisome membrane. Pex5 and
Pex14 form a pore in the membrane and the cargo protein is translocated into the
peroxisome. Pex5 is then recycled to the cytosol.
 Biogenesis of peroxisomes by division.
o New peroxisomes can be formed by the growth and division of old ones, similar
to the division of mitochondria.
o Mediated by the GTPase Drp1
o This is faster.
o De novo formation yields entirely new contents.
o The two pathways both contribute to maintaining the peroxisome population of
the cell by responding to different metabolic needs.
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