Intracellular Compartments and Protein Transport Flashcards by Chris Idone

What is the difference between a nuclear pore complex and a channel?

Nuclear pore complex:
* Small molecules can pass non-selectively
* Much larger than channels
* Nothing regulating ions (large sized pores)
* Large macromolecules enter/exit nucleus selectively

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(1) How are membrane proteins vs. cytosolic proteins vs. secreted proteins translated? (2) How are proteins targeted
to the ER membrane? Hint: what proteins and receptors are involved?

(1)
Membrane proteins:
- N-terminal signal sequence + stop-transfer sequence (sequence of hydrophobic amino acids)

N-terminal signal sequence is cleaved off
Stop-transfer sequence remains in bilayer (forms a-helical membrane spanning segment that anchors protein in membrane)
C-terminal (cytosolic side) continues synthesis

Cytosolic proteins:
- no targeted signals
- get translated in cytosol

Secreted proteins:
- SRP in cytosol binds both ribosome and ER signal sequence as it emerges from ribosome (pauses translation)

Signal peptidase - cleaves off signal sequence (located on ER lumen)
Once C-terminus has passed through
translocator, protein is released into ER lumen

(2)
- Signal recognition particle (SRP) binds to ribosome and ER signal sequence (slows protein synthesis)

SRP receptor (embedded in ER membrane) recognizes SRP
SRP is released and receptor passes ribosome to translocator in ER membrane (translation resumes)

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Explain the requirements and mechanism for protein import into the nucleus. (2) How is this regulated? Hint:
what are the roles of the import receptor, Ran-GTP, Ran-GDP, GEF, and GAP?

Nuclear localization signal (NLS) - signal sequence that directs a protein from cytosol into nucleus; consists of positively charged lysines or arginines

Nuclear import receptors - recognize nuclear localization signal on proteins

(2)
Ran-GTP - present in high concentrations in nucleus

Ran-GDP - present in high concentrations in cytosol

Ran-GEF - causes Ran-GDP to release its GDP and take up GTP; found in nucleus

Ran-GAP - trippers GTP hydrolysis; converts Ran-GTP to Ran-GDP; found in cytosol

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Explain how cargo is selected during clathrin-coated vesicles transport. What is the role of clatherin? Adaptin? Dynamin? SNARES? Rab? (2) How is clatherin functionally different from COPI and II coats?

Cargo interacts with cargo receptor
Clathrin - coats/molds the vesicles
Adaptin - connects cargo receptor to clathrin coat
Dynamin - pinches off vesicle (via GTP hydrolysis)
Rab proteins - direct vesicle to specific spots on correct target membrane
Tethering proteins - recognize and bind to Rab protein on vesicle surface; located at target membrane
SNAREs - catalyze fusion of membranes

(2)
- Clathrin-coated: Golgi -> Lysosomes OR Plasma membrane to endosomes

COPII - ER -> Golgi
COPI - Golgi -> ER

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Explain how a genetic defect involving membrane transport can lead to heart disease (atherosclerosis).

Individuals with defective (mutant) LDL receptors build up cholesterol in blood (can’t be transported into cells) leading to heart disease

i.e., LDL cannot bind to LDL receptor (accumulates in blood)

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Why will molecules in the cytosol not get degraded if a hydrolase from the lysosome accidently leaks out?

Not the correct/optimal pH
Enzymes only work at optimal pH

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What is the relationship between an endosome and a lysosome?

Endosome:
- early precursor compartments
- have pH lower than cytosol (not as low as lysosome)

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(1) What is the glycocalyx? (2) Why is it important? (3) How and where in the cell are proteins glycosylated?

(1) Carbohydrate layer on surface/outside of plasma membrane

(2) Important for protection, recognition, lubrication

(3)
- Where: ER membrane
- Oligosaccharide (14 sugars) is transferred to amino group of an asparagine side chain on protein

Oligosaccharyl transferase - transfers oligosaccharide to target asparagine

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In what ways can the unfolded protein response (UPR) lead to quality control in the ER?

Inhibit protein synthesis
Leads to expansion of ER size
Increase production of chaperones
If cell becomes overwhelmed, then it can self destruct (apoptosis)

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What are the differences between the constitutive and regulated exocytosis pathways?

Constitutive exocytosis:
- Supplies plasma membrane with newly made lipids and proteins
- Does not rely on specific protein signal
- Supplies proteins to ECM
- Enables PM to expand prior to cell division
- Refreshes old lipids and proteins in nonproliferating cells
- Operates continually
- Secretes soluble proteins

Regulated exocytosis:
- Operates only in cells specialized for secretion
- Selected proteins in trans Golgi are diverted into secretory vesicles

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What are examples of protein types that use constitutive exocytosis? Regulated exocytosis?

Constitutive - proteins in ECM (e.g., fibroblasts produce collagen, elastin)
Regulated - hormones, neurotransmitters

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Does exocytosis cause PM to expand indefinitely? Why or Why not?

No - exocytosis usually happens at same time as endocytosis (there is a balance)

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What is the role of Mannose-6-phosphate in protein targeting?

Tag lysosomal enzymes designated for lysosome

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What are the functions of the following membrane-enclosed organelles: cytosol, nucleus, ER, Golgi, Lysosomes, Endosomes, Peroxisomes?

Cytosol -

Nucleus - contains main genome; DNA and RNA synthesis

ER - synthesis of most lipids; synthesis of proteins for distribution to many organelles and to the plasma membrane

Golgi apparatus - modification, sorting, and packaging of proteins and lipids for either secretion or delivery to another organelle

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What are functions of the ER?

protein synthesis
lipid synthesis
Ca2+ store

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What are the three mechanisms in which proteins are transported into organelles?

Transport through nuclear pores
Transport across membranes
Transport by vesicles

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What happens to proteins that lack a sorting signal?

Protein will not go to designated compartment (stay in cytosol)

Accumulate in wrong place

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What are signal sequences? What are some examples?

Signal sequence - amino acid sequence that directs a protein to a specific location in cell

Examples:
- Import into ER: mainly hydrophobic and followed by some negatively charged amino acids

Retention in lumen of ER: “KDEL” sequence
Import into nucleus: positively charged amino acids
Export from nucleus: hydrophobic amino acids
Import into peroxisomes:

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What physical properties of signal sequences are important?

Hydrophobicity
Placement of charged amino acids

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What are examples of protein imported into the nucleus?

DNA/RNA polymerase, exosome

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What are examples of protein exported out of the nucleus?

Cap binding protein, poly A binding protein, exon junction complex

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What two proteins get targeted into the ER?

Into ER and secreted out
Hydrophobic stretch in addition to peptide and get embedded (membrane proteins)

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Describe the nuclear envelope.

composed of an inner and outer membrane (continuous with ER)
support underlying nuclear lamina
contains proteins (inner membrane) that interact with chromatin
perforated by nuclear pores (gates through which molecules enter or leave nucleus)

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What type of proteins make up nuclear lamina?

Intermediate filaments

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How are nuclear import receptors recycled into the cytoplasm?

By interacting w/Ran-GTP

What happens in cells mutant for Ran-GEF? Ran-GAP?

Ran-GEF: - can't charge Ran-GTP - leaves a lot of Ran-GDP (can put a lot of things in but can't put a lot of things out) - Receptor cannot dissociate from cargo (cargo cannot be unleashed) Ran-GAP: - Accumulation of proteins that cannot get inside - Import is disrupted * GEF: protein gets in but cannot dissociate

What signal sequences serves as an import signal for many peroxisomal proteins?

Short sequence of three amino acids

(1) What is zellweger syndrome? (2) What causes it?

(1) Severe abnormalities in brain, liver, and kindeys (2) Mutations that block peroxisomal protein import

The ER serves as a starting point for proteins destined for other organelles. What are some of these organelles?

1. Golgi 2. Endosomes 3. Lysosomes 4. Plasma membrane

What two types of proteins are synthesized on the ER?

1. Water soluble proteins transferred to ER lumen (destined for secretion or residents in an organelle) 2. Prospective transmembrane proteins that become embedded in ER membrane (destined to reside in membrane of organelles or in plasma membrane)

What is the ER signal sequence?

Directs proteins to ER (8+ hydrophobic amino acids)

Compare free ribosomes and membrane-bound ribosomes.

Free ribosomes - unattached to any membrane and make soluble proteins not associated with ER; translate proteins with no ER signal sequence Membrane-bound ribosomes - attached to cytosolic side of ER membrane (make proteins translocated into ER); translate proteins containing ER signal sequence

T or F: proteins enter ER after being synthesized

Proteins enter ER WHILE being synthesized

What are possible destinations for soluble proteins translocated into ER?

1. Can be secreted (secretory pathway) * Happens in endocrine cells 2. “KDEL sequence” * Retention signal * Interacts with KDEL receptor

What is the difference between exocytosis and endocytosis?

Exocytosis: - Vesicle fuses with plasma membrane - Balanced by endocytosis - E.g., neurotransmitters, peptide hormones, digestive enzymes Endocytosis: - Internalizes external components - Extracellular materials are captured by vesicles that bud inward from plasma membrane and are carried into cell - E.g., phagocytes

(1) What is the secretory pathway (exocytosis)? (2) Endocytotic pathway?

(1) Major outward moving system - protein synthesis on ER Membrane -> Golgi apparatus -> plasma membrane OR lysosomes (vis early and late endosomes) (2) Major inward moving system -> plasma membrane -> endosomes -> lysosomes (via late endosomes)

(1) What is clathrin? (2) Describe its structure.

(1) Protein that makes up outer coat of vesicles (2) Form basket-like triskelion cages that help shape membrane into vesicles

What are the two functions of the coat?

1. Helps shape membrane into a bud 2. Captures molecules for onward transport

Describe the process of how clathrin-coated vesicles are formed.

1. Coat assembly and cargo selection * Adaptin - adaptor molecule that connects cargo to clathrin 2. Bud formation 3. Vesicle formation * Dynamin - pinches off vesicles 4. Uncoating * Coat proteins are removed (Clathrin dissociates)

What is the difference between v-SNAREs and t-SNAREs?

v-SNAREs - found on vesicle membranes t-SNAREs - found on target membranes

(1) What is glycosylation? (2) Where does it occur? (3) When does it occur?

(1) Transfer of sugar units to target protein; proteins that enter ER lumen are converted to glycoproteins in ER by covalent attachment of oligosaccharide side chains (2) ER (3) Translation

What is the role of dolichol in glycosylation?

Specialized lipid that attaches oligosaccharide

Misfolded or assembled proteins are retained in ER by binding to ________________.

Chaperone proteins

What are cisternae?

Collection of flattened, membrane-enclosed sacs in Golgi apparatus

What are the two faces of the Golgi? Where are they located?

1. cis (entry) face - adjacent to ER 2. trans (exit) face - points toward plasma membrane

(1) What type of signal sequence on proteins return them back to the ER? (2) What vesicle coat promotes this?

(1) ER retention signal (2) COPI vesicles

(1) What is endocytosis? (2) What are the two types?

(1) Process by which cells take in materials through an invagination of plasma membrane (2) 1. Pinocytosis - ingestion of fluid and molecules; particles <150 nm in diameter) 2. Phagocytosis - ingestion of large particles (e.g., microorganism and debris); particles >250 nm in diameter

What defines early vs late endosomes?

Early endosome - a little acidic Late endosome - more acidic

What is the main difference between the Golgi and endosomes?

Golgi sorts outgoing vesicles Endosome sorts incoming vesicles

How is the interior of the endosome kept acidic?

Through ATP-driven H+ pump (pumps H+ into endosome lumen from cytosol)

What are three fates of receptor proteins following their endocytosis?

1. Degradation - degraded by lysosomes 2. Recycling - receptors return to same plasma membrane domain from which they came 3. Transcytosis - receptors are proceeded to a different domain of plasma membrane

What is receptor-mediated endocytosis?

Mechanism of selective uptake of material in which a macromolecule binds to a receptor

Give and explain an example of receptor-mediated endocytosis.

- LDL bind to LDL receptor on cell surface - LDL receptor enters endosome -> LDL dissociates from its receptor - LDL enters lysosome -> lipoprotein is degraded and free cholesterol is released

What is autophagy?

- cell eats itself - used to degrade obsolete parts of cell - forms autophagosome (fuses with lysosome)

What are the two types of proteins imported into ER?

1. Transmembrane proteins - embedded in ER membrane 2. Water-soluble proteins - fully translocated across ER membrane into ER lumen

What is the ER signal sequence?

Signal sequence that directs proteins to ER membrane

Where is the signal sequence for soluble proteins located?

N-terminus (end that starts synthesis)

What does the cargo receptor do?

Binds adaptin (cytosol) and cargo (lumen)

What is the purpose of the attached oligosaccharides on proteins?

1. Prevent protein degradation 2. Hold protein in ER until it is folded 3. Serve as packaging signal 4. Become part of glycocalyx at PM to promote cell identity

Why is it called N-linked glycosylation?

Oligosaccharide side chains are linked to an asparagine NH2 group

How is pinocytosis different from receptor-mediated endocytosis?

Pinocytosis - indiscriminate and brings in large amounts of fluid Receptor-mediated endocytosis - increases efficiency of specific molecules (less fluid)

What is vesicular transport?

Movement of material between organelles

What triggers the unfolded protein response?

Accumulation of misfolded proteins in ER

What are phagocytic cells? What are some examples?

- cell specialized to take up particles and microorganisms by phagocytosis - e.g., Macrophages, Neutrophils