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

Describe the endoplasmic reticulum

network of membrane-enclosed tubules and sacs (cisternae) extending from the nuclear membrane throughout the cytoplasm

What four cellular components are involved in protein processing and are connected by vesicular transport?

endoplasmic reticulum, golgi apparatus, endosomes, lysosomes

What are newly synthesized proteins labeled with?

radioisotopes

What is a "chase"?

after labeling with radioisotopes proteins are incubated with non-labeled amino acids for different lengths of time

this allows them to track labeled proteins

What is secretory pathway for proteins after incubation with non-labeled amino acids?

ER--> golgi apparatus--> secretory vesicles--> outside cell

What happens to proteins synthesized on free ribosomes? What is this called?

stay in cytosol or transported to nucleus and other organelles

posttranslational translocation

What happens to proteins synthesized on membrane-bound ribosomes? What is this called?

translocated directly into the ER

cotranslational translocation

When cells are disrupted and the nuclei centrifuged out, the ER breaks up into small vesicles called ___.

microsomes

What removes signal sequences when microsomes are added?

proteolytic cleavage

Describe the signal sequence of growth hormones.

roughly 20 amino acids including hydrophobic residues

residues located at amino terminus of polypeptide chain

What binds contranslational targeting and what is it made up of?

signal recognition particle (SRP)

consists of 6 polypeptides and small cytoplasmic RNA called SRP RNA

Describe the process for cotranslational targeting.

-SRP binds to ribosome and signal sequence, translation inhibited

-complex binds to SRP receptor on rough ER membrane

-SRP is released, ribosome binds to membrane channel (translocon)

-signal sequence inserted into translocon and translation resumes

-signal sequence cleaved by signal peptidase and released in ER lumen

Describe posttranslation translocation (common in yeast).

polypeptides synthesized on free ribosomes, signal sequences, receptor proteins on translocon

Proteins are synthesized on ___

free cystolic-bound ribosomes

What receptor protein is associated with the translocon in the ER membrane?

Sec62/63 complex

What acts as a ratchet to pull the polypeptide chain through the channel and into the ER?

BiP: Hsp70 chaperone in the ER

Proteins destined for incorporation into membranes are initially inserted into the ___ instead of being released into the ___

ER membrane

lumen

What makes up the membrane-spanning regions of proteins destined for incorporation into membranes?

alpha helical regions with hydrophobic amino acids

What terminus is on the cystolic side of proteins destined for incorporation into membranes?

amino (N) terminus or carboxy (C) terminus

What is topologically equivalent to the exterior of the cell?

the lumen of the ER

Domains of membrane proteins that are exposed on the cell surface correspond to regions of ___ chains that are translocated into the ___.

polypeptide

ER lumen

What sequences directly insert proteins into the ER membrane? What recognizes these sequences?

internal transmembrane sequences

recognized by SRP, but not cleaved by signal peptidase

How do transmembrane alpha helices exit the translocon? How are the polypeptides oriented across the membrane?

exit translocon laterally and anchor proteins in the ER membrane

oriented in either direction

What signal sequence and middle section do some proteins have that halts translocation and anchors the polypeptide in the membrane? What portion of the growing polypeptide remains in the cytosol?

amino terminal signal sequence

transmembrane and an alpha helix in the middle

carboxy terminal portion

Proteins that span the membrane multiple times are inserted as a result of what?

series of transmembrane sequences with alternating orientations

In what two instances can protein folding and processing occur?

during translocation across the ER membrane or within the ER lumen

What are the two primary roles of lumenal ER proteins?

assist folding and assemble newly translocated polypeptides

How does the Hsp70 chaperone BiP work with an unfolded polypeptide chain?

binds to unfolded polypeptide chain as it crosses the membrane and mediates folding and assembly of multisubunit proteins

Formation of ___ bonds is important in protein folding.

disulfide

Is the cytosol a reducing or oxidizing environment? What happens here?

reducing environment

cysteine residues are in reduced (-SH) state

Is the ER a reducing or oxidizing environment? What happens here and what facilitates this?

oxidizing environment

promotes disulfide bond formation (S-S)

facilitated by protein disulfide isomerase

What are proteins glycosylated on as they're translocated into the ER?

specific asparagine residues (N-linked glycosylation)

The ___ is synthesized on a ___ carrier.

oligosaccharide

lipid (dolichol)

What functions does glycosylation serve?

prevents protein aggregation in the ER and provides signals for subsequent sorting

What attaches some proteins to the plasma membrane? Where are they assembled and where to they attach?

glycolipids called glycosylphosphatidylinositol (GPI) anchors

assembled in the ER membrane

added to carboxy terminus of polypeptide

How are misfolded proteins removed from the ER? What happens to them?

removed by ER-associated degradation (ERAD)

they're identified, returned to the cytosol, degraded by ubiquitin-proteasome system

What acts as sensors of misfolded proteins?

chaperones and protein processing enzymes in ER lumen

Describe the pathway of protein folding that involves the chaperone calreticulin.

-protein folding sensor passes correctly folded glycoproteins to the transitional ER

-folding sensor adds a glucose residue to a misfolded protein and cycles it back to calreticulin to re-attempt folding

-EDEM1 recognizes severely misfolded glycoproteins, removes mannose residues, returns to cytosol through ubiquitin ligase complex where it's marked by ubiquitylation and degraded in a proteasome

What signaling pathway is activated if an excess of unfolded proteins accumulates?

unfolded protein response pathway (UPR)

Describe the process of the UPR pathway.

-expands the ER and produces more chaperones

-if protein folding can't be adjusted to a normal level, the cell undergoes programmed cell death

What three receptors in the ER membrane are activated by unfolded proteins? What does each do?

IRE1: cleaves pre-mRNA of transcription factor XBP1 and activates it

XBP1: translocates to the nucleus and stimulates transcription of UPR genes

ATF6: cleaved to release active ATF6 transcription factor

What protein kinase phosphorylates translation factor eIF2? What does this do?

PERK

inhibits general translation and reduces the amount of protein entering the ER

How are hydrophobic membrane lipids synthesized?

synthesized in association with already existing membranes instead of in the aqueous cytosol

What three lipids make up eukaryotic membranes?

phospholipids, glycolipids, cholesterol

Where are phospholipids synthesized and from what?

synthesized on cytosol side of ER membrane from water-soluble precursors (glycerol)

What half of the ER membrane are new phospholipids added to? How are some transferred to the other half when needed?

cytosolic half

transferred through passage of polar head groups through the membrane called flippases

The ER is also the major site of synthesis of ___ and ___. What is the second component converted to and where does this conversion take place?

cholesterol and ceramide

ceramide is converted to glycolipids or sphingomyelin in the Golgi apparatus

The ___ ER is abundant in cells with ___.

smooth

active lipid metabolism

___ are synthesized from cholesterol in the ER.

steroid hormones (abundant smooth ER is found in cells of testis and ovary)

In the ___, smooth ER contains enzymes that metabolize lipid-soluble compounds and enzymes that inactivate some drugs.

liver

How are phospholipids and proteins exported from the ER and moved to the Golgi apparatus?

exported in vesicles that bud from a specialized region of the ER called the ER exit site (ERES)

vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC)

move to the Golgi apparatus

Proteins in the ___ of one organelle are packaged into ___, then released to the ___ of the recipient organelle following ___.

lumen

budding transport vesicles

lumen

vesicle fusion

T/F: Topological orientation is not maintained during membrane protein and lipid transportation.

F

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

peptide and carbohydrate

default pathway

Where are proteins that function within the ER recognized to be transported back to the ER? What targeting sequence directs retrieval back to the ER?

recognized in the ERGIC or Golgi

targeting sequence KDEL or KKXX at the carboxy terminus

In the Golgi appartus, proteins from the ER are processed and sorted for transport to where (4 options)?

endosomes, lysosomes, plasma membrane, secretion

Most ___ and ___ are synthesized in the Golgi.

glycolipids and sphingomyelin

Describe the structure of the Golgi apparatus.

flattened membrane-enclosed sacs called cisternae and associated vesicles

Where do proteins enter the Golgi apparatus and where do they exit after they're transported through?

enter at the convex cis face (entry face)

exit from the concave trans face (exit face)

What are the four regions of the Golgi apparatus and what does each do?

cis compartment: receives molecules from ERGIC

medial and trans compartments: modifications

trans-Golgi network: sorting and distribution center

Describe the stable cisternae model of Golgi protein movement

proteins carried between cisternae in transport vesicles

Describe the cisternal maturation model of Golgi protein movement

proteins carried within cisternae, gradually mature and progressively move through in the cis to trans direction

What portions of glycoproteins are extensively modified in the Golgi?

carbohydrate portions

How are N-linked oligosaccharides added in the ER modified?

by a sequence of reactions catalyzed by enzymes in different compartments

What is O-linked glycosylation?

carbohydrates added to side chains of serine and threonine

What is involved in processing of proteoglycans?

addition of 100 or more carbohydrate chains to a polypeptide that is further modified by addition of sulfate groups

___ and ___ are synthesized from ceramide in the Golgi. ___ is synthesized by transfer of a phosphorylcholine group from phosphatidylcholine to ceramide. Addition of ___ to ceramide yields a variety of different glycolipids.

Glycolipids

sphingomyelin

sphingomyelin

carbohydrates

Where in the Golgi network are molecules sorted and packaged into transport vesicles?

trans Golgi network

What are the three routes that proteins can be transported from the Golgi to the cell surface?

1. direct transport to the plasma membrane

2. recycling endosomes

3. regulated secretory pathways

Regulated pathways induce release of what from endocrine and nerve cells?

endocrine cells: release of hormones

nerve cells: release of neurotransmitters

Proteins aggregate in the trans-Golgi network and are packaged in secretory granules. What ends the storage in granules?

signals direct fusion with the plasma membrane

What are the three families of vesicle coat proteins? What does each do?

COPII-Coated Vesicles: carry proteins from ER to ERGIC then to Golgi

COPI-Coated Vesicles: bud from ERGIC or Golgi and carry cargo back, returns proteins to earlier compartments

Clathrin-Coated Vesicles: transports in both directions between the trans Golgi, endosomes, lysosomes, and plasma membrane

What regulates formation of clathrin-coated vesicles?

regulated by small GTP-binding proteins ARF1 and Sar1 related to Ras and Ran

GTP-binding proteins recruit adap

What are the two domains for plasma membranes in polarized cells of epithelial tissues?

apical and basolateral domains

What two cell types lack lysosomes? Where are proteins transported to from the Golgi? What directs this transport?

yeasts and plant cells

transported from Golgi to vacuole

short peptide sequences

What are the functions of vacuoles?

same as lyososmes and nutrient storage and turgor pressure maintenance

The selectivity of ___ is key to maintaining the functional organization of a cell.

vesicular transport

Describe synaptic transmission.

specialized form of regulated secretion

synapse: junction of neuron with another cell

chemical neurotransmitters are stored in the neuron in synaptic vesicles

What do transport vesicles do after they fuse with the target membrane?

empty their cargo and insert membrane proteins into the target membrane

What two things must happen for fusion of a transport vesicle with its target to happen? What mediates this interaction?

1. vesicle recognizes the correct target membrane

2. vesicle and target membrane fuse and deliver contents to target organelle

mediated by tethering factors and small-GTP binding proteins (Rab proteins)

What are vesicle Rab proteins?

active GTP-bound state, tethering factors

Describe how transmembrane proteins called SNAREs interact in the mechanism of vesicular transport.

-SNARE-SNARE pairing destablizes them so they can fuse

-have central coiled-coil domain that binds to other domains and zips SNAREs

-direct contact and fusion of lipid bilayers

Describe the five steps in the mechanism of vesicular transport.

1. initiate fusion, Rab/GTP on transport vesicle interact with effector proteins and v-SNAREs to assemble pre-fusion complex

2. different Rab protein on target membrane organizes other effector proteins and t-SNAREs

3. effector proteins link membranes by protein-protein interactions (tethering)

4. tethering stimulates Rab/GTP hydrolysis to bring coiled-coil domains of SNAREs together

5. after membrane fusion, NSF/SNAP protein complex disassembles SNAREs allowing reuse

What are lysosomes?

membrane-enclosed organelles that contain enzymes to break down all types of biological polymers

What is Gaucher disease?

enzyme deficiency that prevents hydrolysis of glucosylceramide to glucose and ceramide

What is the end result of lysosomal storage diseases?

accumulation of undegraded materials

What does it mean that most lysosomal enzymes are acid hydrolases?

active at pH of 5 in lysosomes, but inactive at pH 7.2 in cytoplasm

How do lysosomes maintain acidic pH?

proton pump in lysosomal membrane transports protons into the lysosome

How do lysosomes digest material taken up from outside the cell?

endocytosis

What are the three types of endosomes? What does each do?

Early: receive endocytic vesicles from plasma membrane

Recycling: separate molecules targeted for recycling from those destined for degradation, recylced are taken back to plasma membrane

Late: take in molecules destined for degradation

How are mature lysosomes formed?

transport vesicles carrying acid hyrolases from trans-Golgi fuse with late endosomes

___ are targeted to late endosomes by mannose-6-phosphate residues, which are recognized by mannose-6-phosphate receptors in the trans Golgi network and packaged into clathrin-coated vesicles.

acid hydrolases

What is phagocytosis?

specialized cells (ie macrophages) take up and degrade large particles (including bacteria, cell debris, aged cells)

particles are taken up in vacuoles called phagosomes and fuse with lysosomes to become phagolysosomes

What is autophagy?

turnover of cell's own components

small area of cytoplasm or organelle enclosed in vesicle called autophagosome that fuses with lysosome and contents are digested

What triggers autophagy?

nutrient starvation: degrade nonessentail macromolecules so components can be reutilized

also important for programmed cell death