Textbook: Chapter 8: ER/Golgi Flashcards
1
Q
Some enveloped viruses, such as the herpes virus, fuse directly with the plasma membrane, depositing the genome-laden capsid inside the cell. A larger proportion of viruses, however…
A
hijack endocytic pathways to enter the cell, thereby leaving few traces on the cell surface to alert the attention of roving immune cells. In these cases, viruses are internalized into vesicles known as endosomes and later release their genome into the cell.
2
Q
endomembrane system includes
A
endoplasmic reticulum, Golgi complex, endosomes, lysosomes, and vacuoles
3
Q
peroxisomes
A
organelles that sequester diverse oxidative reactions
4
Q
Transport vesicles move through the cytoplasm in a directed manner, often …….. When it reaches its
destination,…….
A
- pulled by motor proteins that operate on tracks formed by microtubules and microfilaments of the cytoskeleton
- a vesicle fuses with the membrane of the acceptor compartment, which receives the vesicle’s soluble cargo as well as its membranous wrapper
5
Q
Materials follow the biosynthetic (or secretory) pathway from the endoplasmic reticulum, through the Golgi complex, and out to various locations including (6):
A
lysosomes, endosomes, secretory vesicles, secretory granules, vacuoles, and the plasma membrane.
6
Q
endocytic pathway organelle order:
A
Materials follow the endocytic pathway from the cell surface to the interior by way of endosomes and lysosomes, where they are generally degraded by lysosomal enzymes.
7
Q
constitutive secretion (2)
What it is+ contribites to
A
- materials are transported in secretory vesicles from their sites of synthesis and discharged into the extracellular space in a continual manner.
- Most cells engage in constitutive secretion, a process that contributes not only to the formation of the extracellular matrix, but also to the formation of the plasma membrane itself.
8
Q
regulated secretion + ex (2)
A
- materials are stored as membrane-bound packages and discharged only in response to an appropriate stimulus.
- Regulated secretion occurs, for example, in endocrine cells that release hormones, in pancreatic acinar cells that release digestive enzymes, and in nerve cells that release neurotransmitters
9
Q
Proteins, lipids, and complex polysaccharides are transported through the cell along the —- or —- pathway
A
- biosynthetic
- secretory
10
Q
Biosynthetic organell order:
A
- A biosynthetic pathway can be discerned in which
proteins are synthesized in the endoplasmic reticulum, modified during passage through the Golgi complex, and transported from the Golgi complex to various destinations, such as the plasma membrane, a lysosome, or the large vacuole of a plant cell
11
Q
These various types of cargo—secreted proteins, lysosomal enzymes, and membrane proteins—are routed to their appropriate cellular destinations by virtue of specific “addresses” or sorting signals. Explain how this works:
A
sorting signals that are encoded in the amino acid sequence of the proteins or in the attached oligosaccharides. The sorting signals are recognized by specific receptors that reside in the membranes or surface coats of budding vesicles, ensuring that the protein is transported to the appropriate destination.
12
Q
Autoradiography provides a means to visualize biochemical processes by allowing …..
A
an investigator to determine the location of radioactively labeled materials within a cell.
13
Q
Autoradiography steps (4):
A
- To determine the sites where secretory proteins are synthesized, slices of pancreatic tissue are incubated in a solution containing radioactive amino acids for a brief period of time. During this period, labeled amino acids were taken up by the living cells and incorporated into the digestive enzymes as they were being synthesized on ribosomes.
- The tissues were quickly fixed, and the locations of proteins that had been synthesized with labeled amino acids during the brief incubation were determined autoradiographically. Using this approach, the endoplasmic reticulum was discovered to be the site of synthesis of secretory proteins
- After incubating the tissue for a brief period in radioactive amino acids, they washed the tissue until it was free of excess isotope and transferred it to a medium containing only unlabeled amino acids.
- the movements of newly synthesized molecules can ideally be followed by observing a wave of radioactive material moving through the cytoplasmic organelles of cells from one location to the next
14
Q
What Pulse and Chase refers to
A
The pulse refers to the brief incubation with radioactivity during which labeled amino acids are incorporated into protein. The chase refers to the period when the tissue is exposed to the unlabeled medium, a period during which additional proteins are synthesized using nonradioactive amino acids
15
Q
The longer the chase, the ……
A
farther the radioactive proteins manufactured during the pulse will have traveled from their site of synthesis within the cell.
16
Q
rough endoplasmic reticulum (RER) (5)
presence of+composed of+continous+the synthesis of+starting
A
- presence of ribosomes bound to its cytosolic surface
- composed of a network of flattened sacs (cisternae)
- continuous with the outer membrane of the nuclear envelope, which also bears ribosomes on its cytosolic surface
- the synthesis of proteins and the synthesis of most of the lipids of a cell’s membranes
- The RER is the starting point of the biosynthetic pathway
17
Q
smooth endoplasmic reticulum (SER) (4)
lacks+ membrane+extensively developed in+ functions
A
- lacks associated ribosomes
- the membranes of the SER are highly curved and tubular, forming an interconnecting system of pipelines traversing the cytoplasm
- extensively developed in a number of cell types, including those of skeletal muscle, kidney tubules, and steroid-producing endocrine glands
- They sequester calcium ions within the cytoplasm of cells. The regulated release of Ca2+ from the SER of skeletal and cardiac muscle cells (known as the sarcoplasmic reticulum in muscle cells) triggers contraction
18
Q
The two types of ER in terms of their membranes are….
not ribosomes.
A
their membranes are continuous
19
Q
Further experiments revealed that polypeptides are synthesized at two distinct locales within the cell:
A
- on ribosomes attached to the cytosolic surface of the RER membranes
- free ribosomes
20
Q
Proteins synthesized on ribosomes attached to the cytosolic surface of the RER membranes (2):
released into+ process is called+ includes what type of protein?
A
- released into the ER lumen in a process called co translational translocation
- include (1) secreted proteins, (2) integral membrane proteins, and (3) soluble proteins that reside within compartments of the endomembrane system, including the ER, Golgi complex, lysosomes, endosomes, vesicles, and plant vacuoles
21
Q
Protein synthesized on free ribosomes (2):
released in+includes what type of proteins
A
- released into the cytosol
- includes (1) proteins destined to remain in the cytosol (such as the enzymes of glycolysis and the proteins of the cytoskeleton), (2) peripheral proteins of the cytosolic surface of membranes (such as spectrins and ankyrins that are only weakly associated with the plasma membrane’s cytosolic surface), (3) proteins that are transported to the nucleus, and (4) proteins to be incorporated into peroxisomes, chloroplasts, and mitochondria.
22
Q
Secretory proteins contain a signal sequence at their ……. that….
A
- N-terminus
- directs the emerging polypeptide and ribosome to the ER membrane.
23
Q
Co-translational translocation
signal sequence:
A
a stretch of 6–15 hydrophobic amino acid residues
24
Q
Steps for Co-translational translocation (10)
A
- Begins on a free ribosome
- Signal sequence (directs ribosome to membrane) usually at N-terminal end is 6-15 hydrophobic amino acids
- Signal secognition particle (SRP) binds to the signal sequence and the ribosome
- Polypeptide synthesis is halted temporarily
SRP directs this complex to the ER membrane by interaction with the SRP receptor (ribosome+peptide) - Ribosome/polypeptide then are transfered from the SRP once it docs to the receptor to the translocon (a protein pore in the ER membrane with a plug to prevent ion diffusion)
- Contact with the signal sequence displaces the plug
SRP released from the SRP receptor - Translocation through the pore: Polypeptie enters the ER lumen
- Upon termination, ribosome is released
- Signal sequence is removed by an enzyme: signal peptidase
- Protein chaperones (Eg: BiP) aid in protein folding
25
GTP- and GDP-bound versions of a G protein
the GTP-bound protein typically turns the process on, and hydrolysis of the bound GTP turns it off
26
The -------- bound to these two proteins ( SRP and the SRP receptor) triggers the release of the signal sequence by the SRP.
hydrolysis of GTP
27
Processing of Newly Synthesized Proteins in the Endoplasmic Reticulum after Co-translational translocation (2)
- The N-terminal portion containing the signal peptide is removed from most nascent polypeptides by a proteolytic enzyme, the signal peptidase
- Carbohydrates are added to the nascent protein by the enzyme oligosaccharyltransferase
28
Both the signal peptidase and oligosaccharyltransferase are .....
integral membrane proteins
29
However, unlike soluble secretory and lysosomal proteins, which pass entirely through the ER membrane during translocation, integral proteins ......
contain one or more hydrophobic transmembrane segments that are shunted directly from the channel of the translocon into the lipid bilayer.
30
Synthesis of Integral Membrane Proteins on ER-Bound Ribosomes steps (3)
1. Synthesized by co-translational translocation using the same machinery as secreted proteins (SRP, receptor etc)
SRP recognizes the hydrophobic transmembrane domain as the signal sequence
2. The nascent polypeptide enters the translocon just as if it were a secretory protein. However, the entry of the hydrophobic transmembrane sequence into the pore blocks further translocation of the nascent polypeptide through the channel.
3. As polypeptide pass through the translocon, a gate in the pore opens and allows the proteins to partition themselves according to their solubility properties (Either in the aqueous pore or in the hydrophobic lipid bilayer
31
Membrane Biosynthesis in the Endoplasmic Reticulum
1. Membranes grow as newly synthesized proteins, and lipids are inserted into existing membranes in the endoplasmic reticulum (ER).
2. Membrane components move from the ER to virtually every other compartment in the cell. As the membrane moves from one compartment to the next, its proteins and lipids are modified by enzymes that reside in the cell’s various organelles. These modifications contribute to giving each membrane compartment a unique composition and distinct identity
32
The addition of sugars to an oligosaccharide chain is catalyzed by a large family of membrane-bound enzymes called
glycosyltransferases.
33
The sequence in which sugars are transferred in glycosylation depends on ...
the sequence of action of glycosyltransferases that participate in the process. This in turn depends on the location of specific enzymes within the various membranes of the secretory pathway. Thus, the arrangement of sugars in the oligosaccharide chains of a glycoprotein depends on the spatial localization of particular enzymes in the assembly line
34
N-linked oligosaccharide in the RER steps (7):
1. First 7 sugars are transferred one at a time to a lipid: dolichol pyrophosphate, embedded in the ER membrane
2. Initially assembly is on the cytosolic side
3. Sugars are added by glycosyltranferases
4. Dolichol and attached oligosaccharide is flipped across the membrane
5. These latter sugars are attached one at a time on the cytosolic side of the membrane to the end of a dolichol phosphate molecule, which then flips across the membrane and donates its sugar to the growing end of the oligosaccharide chain
6. Completed oligosaccharide is transfered to an asparagine residue of the polypeptide being translated
7. Transfer by the enzyme oligosaccharyltransferase to an Asn within the sequence: x--x-Asn-X-ser/thr
35
The basal, or core, segment of each carbohydrate chain is not assembled on the protein itself but put together independently on a lipid carrier and then transferred, as a block, to specific asparagine residues of the polypeptide. This lipid carrier, which is named -----, is embedded in the ---- membrane
- dolichol phosphate
- ER
36
Sugars are added to the dolichol phosphate molecule one at a time by membrane-bound -----,
glycosyltransferases
37
This preassembled block of 14 sugars is then transferred by the ER enzyme ------ from dolichol phosphate to certain asparagines in the nascent polypeptide as the polypeptide is being translocated into the ER lumen.
- oligosaccharyltransferase
38
quality control
1. Glucosidase 1 and 2 removes 2 glucoses
2. Glycoprotein with one glucose is recognized by calnexin (chaperone protein in the ER)
3. Calnexin removes another glucose
4. Incompletely folded proteins are recognized by UGGT (a conformation sensing enzyme): detects exposed hydrophobic residues. Adds glucose molecule.
5. *rechecks by calnexin if UGGT adds glucose*
6. Properly folded proteins exit to Golgi
7. Improperly folded proteins are degraded in a proteosome in the cytosol
39
UGGT recognizes incompletely folded or misfolded proteins because they ....
display exposed hydrophobic residues that are absent from properly folded proteins
40
soon after vesicles bud from the ER membrane tell me the steps that happen before reaching Golgi (2) :
1. transport vesicles fuse with one another to form larger vesicles VTCs, and interconnected tubules in the region between the ER and Golgi complex
2. Once formed, the VTCs move farther away from the ER toward the Golgi complex. Movement of VTCs occurs along tracks composed of microtubules.
41
cis Golgi network (CGN) function
- function primarily as a sorting station that distinguishes between proteins to be shipped back to the ER and those that are allowed to proceed to the next Golgi station
42
trans Golgi network (TGN) function
- The TGN is a sorting station where proteins are segregated into different types of vesicles heading either to the plasma membrane or to various intracellular destinations.
43
In the Golgi complex, as in the RER, the sequence in which sugars are incorporated into oligosaccharides is determined by .....
the spatial arrangement of the specific glycosyltransferases that come into contact with the newly synthesized protein as it moves through the Golgi stack
44
Unlike synthesis of N-linked oligosaccharides, which begins in the ER, those attached to proteins by O-linkages are assembled ....
entirely within the Golgi complex
45
cisternal maturation model
- Golgi cisternae formed at the cis face of the stack by fusion of membranous carriers from the ER and ERGIC and that each cisterna physically moved from the cis to the trans end of the stack, changing in composition as it progressed.
- the cisternae progress gradually from a cis to a trans position and then disperse at the TGN
46
vesicular transport model
- cargo (i.e., secretory, lysosomal, and membrane proteins) is shuttled through the Golgi stack, from the CGN to the TGN, in vesicles that bud from one membrane compartment and fuse with a neighboring compartment farther along the stack.
- the cisternae of a Golgi stack remain in place as stable compartments
47
The vesicular transport model acceptance was based largely on the following observations (2):
1. Each of the various Golgi cisternae of a stack has a distinct population of resident enzymes. How could the various cisternae have such different properties if each cisterna was giving rise to the next one in line, as suggested by the cisternal maturation model?
2. Large numbers of vesicles can be seen in electron micrographs to bud from the rims of Golgi cisternae
48
The cisternal maturation model acceptance was based largely on the following observations (2):
As predicted by the cisternal maturation model, when the formation of transport carriers from the ER is blocked by either treatment of cells with specific drugs or the use of temperature-sensitive mutants, the Golgi complex simply
disappears. When the drugs are removed or the mutant cells are returned to the permissive temperature, the Golgi complex rapidly reassembles as the ER-to-Golgi transport is renewed.
49
Protein coats have at least two distinct functions:
(1) They act as a mechanical device that causes the membrane to curve and form a budding vesicle
(2) they provide a mechanism for selecting the components to be carried by the vesicle
50
COPII-coated vesicles mediate transport from the -----
ER to the ERGIC and Golgi complex.
51
COPI-coated vesicles transport proteins from ..... (2)
- the ERGIC and Golgi complex to the ER
- also transport Golgi enzymes between cisternae in a retrograde direction.
52
Clathrin-coated vesicles mediate transport from....
the TGN to endosomes and lysosomes
53
COPII-Coated Vesicles-Transporting Cargo from the ER to the Golgi Complex steps (7):
1. vesicles, which have been observed to bud off at specialized domains of the ER, are referred to as ER exit sites (ERESs)
2. Sar1-GDP molecules have been recruited to the ER membrane by a protein called a GEF (guanine-exchange factor), which catalyzes the exchange of the bound GDP with a bound GTP
3. each Sar1-GTP molecule has extended a fingerlike α helix along the membrane within the cytosolic leaflet. This event expands the leaflet and induces the curvature of the lipid bilayer at that site
4. a dimer composed of two COPII polypeptides (Sec23 and Sec24) has been recruited by the bound Sar1-GTP. The Sec23–Sec24 heterodimer is thought to further induce the curvature of the membrane in the formation of a vesicle
5. Transmembrane cargo accumulates within the forming COPII vesicle as their cytosolic tails bind to the Sec24 polypeptide of the COPII coat. It interacts with the ER export signals of membrane proteins.
6. the remaining COPII polypeptides (Sec13 and Sec31) have joined the complex to form an outer structural scaffold of the coat.
7. Disassembly is triggered by hydrolysis of the bound GTP to produce a Sar1-GDP subunit, which has decreased affinity for the vesicle membrane. Dissociation of Sar1 GDP from the membrane is followed by the release of the other COPII subunits.
54
COPII coats select and concentrate certain components for transport in vesicles. Certain integral membrane proteins of the ER are selectively captured because they contain “ER export” signals as part of their cytosolic tail. These signals interact specifically with COPII proteins of the vesicle coat. Proteins selected by COPII-coated vesicles include ..... (3)
(1) enzymes that act at later stages in the biosynthetic pathway, such as the glycosyltransferases of the Golgi complex
(2) membrane proteins involved in the docking and fusion of the vesicle with the target compartment
3) membrane proteins that are able to bind soluble cargo (such as the secretory proteins). Cells lacking a specific cargo receptor typically fail to transport a specific subset of proteins from the ER to the Golgi complex
55
The COPI coat is made up of a complex, called -----, made up of ---- proteins.
- coatamer
- seven
56
Studies suggest that proteins are maintained in an organelle by a combination of two mechanisms:
57
Proteins that normally reside in the ER, both in the lumen and in the membrane, contain short amino acid sequences at their ---terminus that serve as retrieval signals, ensuring their return to the ER if they should be accidentally carried forward to the ERGIC or Golgi complex.
- C
58
Soluble resident proteins of the ER lumen (such as protein disulfide isomerase and the molecular chaperones that facilitate folding) typically possess the
retrieval signal “---------” (or ---- in single-letter nomenclature).
- lys-asp-glu-leu
- KDEL
59
KDEL receptor (2)
| Location/what they are+ what they do
- an integral membrane protein that shuttles between the cis Golgi and ER compartments
- recognize KDEL proteins and return them to the ER
60
Membrane proteins that reside in the ER also have a retrieval signal that binds to the -----, facilitating their return to the ER. The most common retrieval sequences for ER membrane proteins is .......
- COPI coat
- KKXX (where K is lysine and X is any residue).
61
Lysosomal proteins are synthesized on ----- and carried to the Golgi complex along with other types of proteins
membrane-bound ribosomes of the ER
62
What happens to Lysosomal proteins once it reaches the Golgi complex?
Soluble lysosomal enzymes are specifically recognized by enzymes that catalyze the addition of a phosphate group to certain mannose sugars of the N linked carbohydrate chains
63
Lysosomal enzymes carrying a ----- signal are recognized and captured by ------ which are integral membrane proteins that span the ---- membranes
- mannose 6-phosphate
- mannose 6-phosphate receptors (MPRs)
- TGN
64
Steps for taking lysosomal enzymes from TGN to lysosome (5)
| extracellular?
- They are selectively incorporated into a clathrin-coated vesicle at the TGN
- The mannose 6-phosphate receptors are thought to have a dual role: They interact specifically with the lysosomal enzymes on the luminal side of the vesicle, and they interact specifically with adaptors on the cytosolic surface of the vesicle.
- The mannose 6-phosphate receptors separate from the enzymes and are returned to the Golgi complex
- The lysosomal enzymes are delivered to an endosome and eventually to a lysosome.
- Mannose 6-phosphate receptors are also present in the plasma membrane, where they capture lysosomal enzymes that are secreted into the extracellular space and return the enzymes to a pathway that directs them to a lysosome
65
The structure of clathrin-coated vesicles/how they bud and form (2):
- G-protein Arf1-GTP binds to the membrane and initiates formation of the budding vesicles and binding of other coat proteins (induces memberane curvature when bound to GTP).
- GGA adaptor molecule then binds to Arf1-GTP, clathrin and cytosolic tails of MPR receptors
66
Lysosomal enzymes are escorted from the TGN by a family of adaptor proteins called -----.
GGAs
67
GGA molecule has several domains, each capable of grasping a different protein involved in vesicle formation (3):
1. The outer ends of the GGA adaptors bind to clathrin molecules, holding the clathrin scaffolding onto the surface of the vesicle.
2. On their inner surface, the GGA adaptors bind to a sorting signal in the cytosolic tails of the mannose 6-phosphate receptors. The MPRs, in turn, bind to soluble lysosomal enzymes within the vesicle lumen.
3. bind to Arf1 and to the surrounding cytosolic network of clathrin molecules
68
After the vesicle has budded from the TGN, ...
| clathrin coat+MPRs
the clathrin coat is lost and the uncoated vesicle proceeds to its destination, which may be an early endosome, late endosome, or plant vacuole. Before they reach one of these organelles, the MPRs dissociate from the lysosomal enzymes and return to the TGN
69
The targeted delivery of integral proteins to the plasma membrane appears to be based largely on ---- in the cytoplasmic domains of the membrane proteins.
sorting signals
70
Targeting Vesicles to a Particular Compartment steps (4):
1. Movement of the vesicle toward the specific target compartment. In many cases, membranous vesicles must move considerable distances through the cytoplasm before reaching their eventual target. These types of movement are mediated largely by microtubules and their associated motor proteins, which act analogously to a railroad system carrying cargo along defined pathways to predetermined destinations.
2. Tethering vesicles to the target compartment. The initial contacts between a transport vesicle and its target membrane, such as a Golgi cisterna, are thought to be mediated by a diverse collection of “tethering” proteins.
3. Docking vesicles to the target compartment. V snare put onto transport vesicles during budding attaches to t snare located in the target membrane.
4. Fusion between vesicle and target membrane: interactions between t snare and v snare pull lipid bilayers together with enough force to cause fusion
71
Two groups of tethering proteins have been described : ------: fibrous proteins that are capable of forming a molecular bridge between the two membranes over a considerable distance (50–200nm) and large ------: that appear to hold the two membranes in closer proximity
- rod-shaped
- multiprotein complexes
72
small G proteins called Rabs functions (2):
1. play a key role in vesicle targeting by recruiting specific cytosolic tethering proteins to specific membrane surfaces
2. play a key role in recruiting numerous proteins involved in other aspects of membrane trafficking, including the motor proteins that move membranous vesicles through the cytoplasm
73
v-SNAREs
become incorporated into the membranes of transport vesicles during budding
74
t-SNARE
located in the membranes of target compartments
75
As the synaptic vesicle and presynaptic membrane approach one another, the SNARE motifs of t- and v-SNARE molecules from apposing membranes interact to form .....
four-stranded bundles
76
According to current consensus, the ability of a particular vesicle and target membrane to fuse is determined by the specific combination of interacting proteins —including -------- that can be assembled at that site in the cell. Taken together, these multiple interactions among several types of proteins provide a high level of specificity, ensuring that each membrane compartment can be selectively recognized.
tethering proteins, Rabs, and SNAREs
77
The enzymes of a lysosome share an important property: All have their optimal activity at an acid pH and thus are acid hydrolases. The pH optimum of these enzymes is suited to the low pH of the lysosomal compartment, which is approximately ----.
- 4.6
78
autophagy
regulated destruction and replacement of the cell’s own organelles
79
autophagy steps:
An organelle, such as the mitochondrion, is surrounded by a doublemembrane structure to produce a double-membrane sequestering compartment called an autophagosome. In mammalian cells, autophagosomes. Once formed, the outer membrane of the autophagosome fuses with a lysosome, generating a structure called an autolysosome, in which both the inner
membrane of the autophagosome and the enclosed contents are degraded. The products of these degradative reactions are made available to the cell.
80
autophagosome
organelle surrounded by a double membrane structure
81
Bulk-phase endocytosis (also known as pinocytosis)
the nonspecific uptake of extracellular fluids. Any molecules, large or small, that happen to be present in the enclosed fluid also gain entry into the cell.
82
receptormediated endocytosis (RME)/ clathrin-mediated endocytosis +ex
brings about the uptake of specific extracellular macromolecules (ligands) following their binding to receptors on the external surface of the plasma membrane (ex: hormones, growth factors, nutrients)
83
Ap2
| clathrin-mediated endocytosis
links cytoplasmic tails of plasma membrane receptors with clathrin
84
Dynamin (2)
| Acts as a+ what it does
- acts as an enzyme capable of utilizing the chemical energy of GTP to generate mechanical forces.
- Subunits polymerize to form a ring
