BSC 300 Exam 3

Endoplasmic Reticulum (ER)

Comprises a network of membranes that penetrates much of the cytoplasm.

Divided into Smooth and Rough

Rough ER (RER)

Is continuous with the outer membrane of the nuclear envelope.

Composed of a network of flattened sacs called cistenae

Attached ribosomes actively synthesize proteins

Smooth ER

Synthesis of steroid hormones in endocrine cells.

Detox the liver

Sequestration of calcium ion from cytoplasm of muscle cells (sarcoplasmic reticulum)

Synthesis of membrane lipids (cholesterol)

Free ribosomes protein synthesis

Proteins can be synthesized on free cytosolic ribosomes, on the nonsecretory pathway

Bound ribosomes protein synthesis

Protein synthesis can occur on ribosomes bound to the ER, on the Secretory pathway.

Protein synthesis BEGINS on the cytoplasmic free ribosomes, and then enter the ER after reading the ER signal sequence

The ER is the entry point for proteins destined for…

Golgi, endosomes, lysosomes, plasma membrane, and for export/secretory proteins

ER signal sequence

Connected to the ribosome and tRNA, that directs the free ribosomes to dock on the ER membrane

Polyribosome complexes

Where both free and bound ribosomes translate mRNA.

Is a single mRNA bound by multiple ribosomes at various stages of translation.

Signal Recognition Particle (SRP) for ER

ER signal sequence on N-terminus is bound by a cytoplasmic protein called the SRP.

SRP delivers the ribosome/mRNA/peptide complex to the SRP receptor on the membrane of the RER - SRP is released

SRP structure breakdown

Cytosolic ribonucleoprotein particle = 6 proteins bound to RNA scaffold

Signal Sequence Binding Domain (P54 subunit in eukaryotes) = M domain methionine and other hydrophobic amino acid in cleft residues binds to the signal sequence hydrophobic core

GTP and receptor binding domain = structure of GTP bound inbetween FtsY and Ffh subunit

FtsY and Ffh subunit binding

They are bound together by GTP binding

This forms two active sites for hydrolysis of both bound GTP molecules (pseudosymmetric dimer).

This brings the ribosome to the ER.

Hydrolysis to GDP causes SRP receptor to dissociate

Translocon

Protein translocator - sits in the plasma membrane, where the ribosome is bound to so that the nascent protein can elongate into the lumen.

Preserves integrity of the ER membrane by two gating mechanisms

Sec61ꭤ translocon complex structure

Lets Nascent secretory proteins pass through its channel (the plasma membrane) into the ER lumen as it elongates.

Hourglass-shaped channel through the center of the pore.

Can be plugged by a helical plug (gating mechanism)

Negative for closed, Positive for open

How does the Nascent protein enter the lumen when bound to the translocon?

The protein is growing into the ER, not pulled.

Translocation through the translocon (Sec61ꭤ) is driven by translation elongation

Cotranslational translocation

Where translation and translocation occur simultaneously.

The protein elongation is occurring as it enters the lumen.

What side does the signal sequence emerge from?

The N terminus side (NH3) during Nascent protein synthesis.

Post-Translational translocation

Some yeast secretory proteins enter the ER lumen through the Sec61 translocon after translation has been completed (no SRP/receptor involvement)

Uses successive cycles of BiP protein chaperone binding to ATP to get through the translocon complex.

ATP hydrolysis (requires additional energy)

Integral membrane protein qualities:

Rough ER synthesizes 5 types.

Are classified by the orientation of the N and C termini in the membrane.

Hydrophobic alpha helices span the membrane bilayer.

Hydrophilic regions are outside the membrane on either side.

Type I ER membrane proteins

There is a Signal Sequence added off the N-terminus on the lumen side.

Also contains a Stop-Transfer Anchor (STA) sequence that creates the middle part that stays in the membrane. Synthesis continues, but it does not elongate into the lumen, it stays in the cytosol until stop codon.

Type II ER membrane protein

Does not have a Signal Sequence, but does have the Signal-Anchor Sequence (SA) - part that stays in membrane

the N-terminus is the Cytosol

When it starts synthesizing, the N-terminus loops back around into the cytosol, and the C-terminus ends up in the lumen,

Type III ER membrane protein

Does not have a Signal Sequence, but does have the Signal-Anchor Sequence (SA) - part that stays in membrane

Difference with Type II, is that the N-terminus is the Lumen.

Elongates as normal from the cytosol into lumen, but most of the chain is in the cytosol

Type IV ER membrane protein

Contains alternating SA and STA sequences as it dips into and out of the membrane.

In Alpha proteins = N-terminus is in the cytosol

In Beta proteins = N-terminus is in the lumen

GPI-anchored proteins

Glycosylphosphatidylinositol molecule is from yeast.

Two portions, a hydrophobic and hydrophilic that sit in their perfered climates and have a protein chain.

Hydropathy profile

predicts likely topogenic sequences in integral membrane proteins.

The spikes in the graph indicate the Stop-transfer sequences.

Oligosaccharide percursor

A preformed N-linked Oligosaccharide is added to many proteins in the rough ER.

Attached on the Asparagine residues.

Dislocation

Misfolded secretory proteins that are recognized by specific ER membrane proteins and targeted for transport from the ER lumen into the cytosol for degradation.

Are marked by N-linked oligosaccharides.

Lectins = Calnexin (CNX) and Calreticullun (CRT)

Used when N-linked Oligosaccharides are used to mark proteins for degradation, they give the proteins more time to fold.

(Retain protein in the ER for folding chaperone activity until properly folded)

Protein Disulfide Isomerase (PDI)

Forms and rearranges proteins and helps them form cysteine disulfide bonds.

Also helps to break misformed disulfide bonds and make the right one.

Transient BiP

It is a chaperone that binds to the Nascent protein as it enters the ER.

This helps prevent premature folding into incorrect conformation.

Unfolded- protein response (UPR)

presence of unfolded proteins in the rough ER increases transcription of genes that encode ER chaperones and other folding catalysts.

Uses Ire I dimer

Nuclear pores

Selective gates into the nucleus.

General Signal Sequence purpose

A sorting signal composed of a unique amino acid sequence that is recognized by other cellular proteins and used as a mailing address for delivery or retention.

Usually cleaved from the mature protein once it reaches its destination.

Proteins for Mitochondrial and Chloroplast matrix

Synthesized on Cytosolic ribosomes and maintain in an unfolded state by chaperones till it enters.

Have N-terminal organelle-specific signal sequence.

TOM complex (Transporter Outer Membrane)

Is a General Import pore, where the signal sequence enters the Outer Membrane.

Protein needs to be unfolded, and uses Cytosolic Hsp70 chaperone to guide it.

TIM complex (Transporter Inner Membrane)

After entering the Intermembrane space through the TOM complex, it goes through the TIM pore - through the Inner Membrane, into the Matrix.

Protein needs to be unfolded.

Matrix Chaperone

Protein that binds the polypeptide chain, and helps it refold once it enters the matrix through the TIM complex.

Cytosolic Hsp 70

The chaperone protein in the cytosol that guides the precursor protein to the TOM complex.

Uses ATP energy to maintain the precursor protein in its unfolded state

Matrix Hsp70

Acts as a molecule motor to pull the protein through the TIM complex, into the matrix.

Transport proteins to Chloroplast Thylakoids

All proteins contain an N-terminal stromal-import sequence.

Requires unfolded precursor proteins

TOC complex

Translocon for Outer membrane of chloroplasts.

Requires ATP energy

TIC complex

Translocon for Inner membrane of chloroplasts.

Requires ATP energy.

SRP-dependent pathway for Cholorplast include… (signal recognizing particle)

Plastocyanin Precursor protein enters the Outer and inner membrane through Toc and Tic complexes.

Sgnal sequence is cleaved off, and SRP is used to guide the chain to the Thylakoid membrane translocon.

Enters the Lumen

pH-dependent pathway for Chloroplast include… (pmf-dependent)

Metal-binding Precursor enters the Outer and Inner membrane through the Toc and Tic complexes.

Signal Sequence is cleaved off, and a Thylakoid targeting sequence (twin arginine) is uncovered. Metal binding proteins partially fold it in the Stroma.

Enters the Thylakoid membrane through a custom pore, driven by Proton-motive force)

When in the lumen, the RR sequence is dissociated.

Peroxisome luminal proteins

Protein chain is sythesized on free ribosomes in the cytosol.

Contains a C-terminal peroxisomal-targeting sequence (PTS1)

PTS1 targeting seqeunce

C-terminal peroxisomal-targeting sequence is tagged on to the end of the Polypeptide chain, and is recognized by Cytosolic receptor (Pex5), that drags it into the peroxismal matrix.

Pex5 receptor

Cytosolic protein receptor that binds the Polypeptide in the cytosol, and imbeds itself into the membrane, where the protein chain is released into the matrix.

Gets reused by going back into the cytosol using Ubiquitin and ATP.

NPCs (Nuclear Pore Complexes)

Spans both inner and outer membrane of the nuclear envelope.

This is where large complex structures enter through the FG-nucleoporins

FG-nucleoporins

Contain multiple repeats of short hydrophobic sequences (FG-repeats (gel like matrix)), that line the central transporter channel.

Act as sink drainers

Nuclear-Localization Signal (NLS)

Proteins imported INTO the nucleus because they are bound to an Importin.

Nuclear-Export Signal (NES)

Helps export the RanGTP and Importin back into the Cytoplasm after the Cargo has been dropped off.

Importin/Cargo complex

Complex of the NLS receptor (importin) and the protein cargo.

The complex enters the nucleoplasm (inside nucleus), and Ran (GTPase) binds to the Importin, which causes the Cargo to be released (allosteric change)

The Nucleoplasm contains high… (nuclear import)

high levels of Ran GEF.

GEF tells the GDP to turn into GTP, so that it can bind to the Importin

The Cytoplasm contains high… (nuclear import)

high levels of Ran GAP

When the RanGTP and Importin complex enters back into the cytoplasm, GAP tells the GTP to hydrolysis into GDP so that the Importin can be let go. (process starts again)

Ran-Independent Nuclear Export

Uses Heterodimeric NXF1/NXT1 nuclear export receptor and ATP to transport cargo

Endomembrane system components?

Transport vesicles, endoplasmic reticulum, Golgi complex, nuclear envelope, endosomes, and lysosomes

Secretory pathway

Distribution of membrane proteins synthesized by the rough ER to the final destination at the cell surface or in lysosomes.

Proteins in transport vesicles bud off the ER and fuse together to form a new cis-Golgi and then trans-Golgi for Cisternal maturation.

Transported by COPII-coated vesicles

Protein trafficking

Retrograde transport vesicles move proteins to the previous Golgi compartment.

Transported by COPI-coated vesicles

GTP-bound form (GTPase switch)

Turns it “on” to its active conformation.

It can interact with target protein to regulate their activities

GEF stimulates it to release GDP and add GTP

GDP-bound form (GTPase switch)

Turns it “off” to its inactive conformation.

GAP stimulates hydrolysis of GTP to become GDP (off)

Vesicle Budding from donor membrane (components)

Cytosolic Coat Protein complex, forms around the membrane

GTP-binding proteins

Membrane Cargo Proteins

Membrane Cargo-Receptor Protein, which receives soluble cargo protein

vesicle-SNARE proteins

Vesicle Fusion with target membrane:

Coat is gone.

v-SNARE on the vesicle interacts with the t-SNARE on the target membrane to form the SNARE complex.

Their twisting causes vesicle membrane fusion.

COPII-coated vesicles

Moves materials from ER to Golgi complex = Anterograde movement

Uses Sar1 GTPase to embed the structure in the membrane, and gets released when coat is disassembled.

COPI-coated vesicles

Moves materials from Golgi backwards to the ER, or from the trans to cis Golgi = Retrograde Movement.

Uses ARF GTPase.

Clathrin-coated vesicles

Moves materials from the trans-Golgi network to endosomes, lysosomes, and plant vacuoles.

Uses ARF GTPase.

Rab GTPases

Control docking of vesicles on target membranes, where the SNARE complexes can then form so the vesicles can fuse. (Rab-GTP hydrolyzed to Rab-GDP)

Dissociation of SNARE complex by NSF and alpha-SNAP proteins)

cis Golgi Network (CGN)

Entry site for material arriving from ER.

KDEL receptor

recognizes and returns soluble ER proteins based on Lys-Asp-Glu-Leu (KDEL) signal.

KKXX retrieval signal

ER membrane proteins have this Lys-Lys-X-X retrieval signal on their C-terminus which binds to COPI coat

cis-Golgi network to ER retrieval mechanism

Acidic pH promotes receptor KDEL interaction.

Missorted ER-resident protein and KDEL peptide sequence is bound in the Golgi (low pH), and un-binds when its transported to the ER because of the high pH.

Process of Cisternal maturation

cis-Golgi network = cis-cisterna = medial cisternae = trans-cisterna = trans-Golgi network

What does the trans-Golgi do?

It sorts proteins into vesicles targeted for different destinations.

Endocytic pathways

1) Secretory vesicles for Regulated secretion from the cytosol (Exocytosis)

2) Constitutive secretion - goes to a specific membrane out of the cell

3) Transport vesicles to Late endosome then lysosomes (Clathrin coated)

4) Transport vesicles to Lysosome (AP complex)

5) Endocytic Vesicle enters into Golgi and goes Retrograde to ER. (COPI coated)

Regulated Exocytosis pathway

For cells specialized for secretion, like hormones or neurotransmitter producing cells.

Have secretory vesicles that go out with a release signal that SIT ALONG THE PLASMA MEMBRANE (ready for it)

AP complex vesicles

Transports lysosomal enzymes directly from the Golgi destined for Lysosome.

May have clathrin coats

Adaptor protein

Clathrin CANNOT interact with protein directly, it uses adaptor proteins to select cargo

What assembles the Clathrin coat?

Arf GTPases

GTPase Dynamin

Plays an essential role in releasing the Clathrin-coated vesicle from the membrane.

It pinches off the membrane stalk to release the clathrin coated vesicle. Uses GTP hydrolysis.

Mannose 6-phosphate (M6P) residues

Sugar residues are added post-translationally to proteins in the Golgi complex.

Serves as a “adress label” to target proteins for proper delivery.

Function in directing newly synthesized lysosomal enzymes to lysosomes.

Process of trafficking lysosomal enzymes from the trans-Golgi network and cell surface TO lysosomes

1) Clathrin-coated vesicles from trans-Golgi and cell surface contain the cargo.

2) Once in the Cytosol, the coat is dissembled (becomes and uncoated vesicle)

3) The M6P receptor is taken off and enters the Late endosome (bc of low pH)

4) Then goes to Lysosome.

Proteolytic processing of proproteins

Constitutive secreted pathway= Endoprotease severs it into the mature form

Regulated secreted pathway= lots more steps, same thing

Endocytosis

Clathrin-coated pits generate endocytic vesicles that are delivered to endosomes.

Pinocytosis is…

Clathrin-dependent.

Focuses in non-specific uptake of cellular fluids

Used for retrieval of membrane components

Phagocytosis is…

Non-clathrin dependent.

Fusion of membrane forms a phagosome that delivers the invader ultimately to Lysosomes for degradation.

Receptor-mediated endocytosis

Is Clathrin-dependent.

Functions in uptake of specific molecules.

Binds to receptors in cell membrane

Early endosomes

Are located near the periphery of the cell.

It sorts material and sends bound ligands to the late endosomes.

Has a acidic pH for cargo to remove from receptor

Late endosomes

Are near the nucleus, also known as multivesicular bodies (MVBs)

Sends to the Lysosome eventually

Lysosomes

Function in cellular digestion.

Uses acid hydrolases for hydrolysis of almost every type of biological macromolecules.

pH optimum in acid range.

House-keeping receptors

Responsible for uptake of materials that will be used by the cell.

Signaling receptors

Responsible for binding ligands that carry messages that charge the activities of the cell.

Low-density lipoprotein (LDL)

Cells take up lipid from the blood in the form of large, well-defined lipoprotein complexes. Like chloestrol.

LDL particle contains only a single molecule of one type of apolipoprotein (ApoB) wrapped around the outside of the particle.

How are Proteins tagged for endosome degradation?

they are tagged with Ubiquitin at the plasma membrane, and ESCRT complexes mediate the pinching inwardly of budding

Apical (upper) surface of each cell

Packed with finger like microvilli that project into the intestinal lumen

Basal (lower) surface

Adhered to basal lamina (ECM region)

ECM

connecting fibers (collagen) and connective tissue in which large, interdigitating ECM Marcomolecules bind to one another

Cell-adhesion molecules (CAMs)

bind to CAMs on other cells for cell-cell adhesion

Cell-matrix adhesion

adhesion receptors bind to ECM components

Adapter proteins

Directly or indirectly link CAMs to the cytoskeleton (actin or intermediate filaments) and intracellular signaling pathways

Tight Junction

Subjacent to the microvilli

Prevents/regulates diffusion of substrates

Gap Junctions

Cluster od connexion channels

Allow movement of small molecules and ions between cytosols of adjacent cells

Adheren junctions (3 types)

Belt desmosome - all the way around

Desmosomes - link intermediate filaments cytoskeletons between neighboring cells (more localized)

Hemidesmosomes - link intermediate filaments cytoskeleton to basal lamina ECM (facal adhesion have transient interactions)