BIO130 Section 2

Enzymes imported into the peroxisome through a transmembrane protein complex

Peroxisome Enzyme Importation

Contain enzymes for oxidative reactions, detoxify toxins, breakdown fatty acid molecules

Peroxisome

Vesicles

What mediates the exchange of materials between the endoplasmic reticulum, Golgi apparatus and the plasma membrane?

Endoplasmic Reticulum and Golgi Apparatus

Where are the major sites of protein glycosylation in the endomembrane system?

There must be an N-terminal ER signal sequence or an internal start-transfer sequence.

Which of the following statements is correct for a transmembrane protein being translated by an ER-bound ribosome?

Cytosol

Where is a protein in the lumen of the rough endoplasmic reticulum LEAST likely to end up?

Volumes of intracellular compartments will differ for different cell types

Volumes of Intracellular Compartments

Half of the cell volume, protein synthesis and degradation, many metabolic pathways, cytoskeleton

Cytosol

Has membrane bound ribosomes, synthesis of soluble proteins and transmembrane proteins for the endomembrane system

Rough Endoplasmic Reticulum

Phospholipid synthesis, detoxification

Smooth Endoplasmic Reticulum

Discrete structure or subcompartment of a eukaryotic cell that is specialized to carry out a specific function, most are membrane enclosed

Organelles

Nucleus, endoplasmic reticulum, golgi apparatus

Examples of Membrane Bound Organelle

Nucleolus, Centrosome

Examples of Organelles that are not Membrane Bound

Proteins are nuclear-encoded, mRNA arrives in cytoplasm and translation starts on ribosomes in cytosol

Protein Sorting

No signal sequence, default location is cytosol

Cytosolic Protein

Have a sorting signal that is called a signal sequence

Sorted Proteins

Stretch of amino acids in a protein that directs protein to the correct compartment, each signal sequence specifies a specific destination in the cell, for example nucleus, mitochondria, ER, peroxisome

Signal Sequence

Recognize signal sequence and take proteins to their destination

Sorting Receptors

Proteins are nuclear encoded, fully synthesized in cytosol before sorting, proteins are imported folded inside nucleus and peroxisomes, proteins are imported unfolded in mitochrondia and plastids

Post-Translational Sorting

Proteins are nuclear encoded, proteins have ER signal sequence and are associated with ER during protein synthesis in cytosol

Co-translational sorting

Sorted during post-translational sorting, proteins are nuclear encoded, fully synthesized in cytosol, transported folded, targeted by a signal sequence for import into nucleus

Transport through Nuclear Pores

Signal Sequence that directs protein from cytosol into nucleus

Nuclear Localization Signal

Cytosolic proteins that recognize the nuclear localization signal, and direct protein to a nuclear pore

Nuclear Import Receptors

Sorted during post-translational sorting, proteins are nuclear encoded and folded during transport

Sorting Proteins to Peroxisome

Sorted during post-translational sorting, proteins are nuclear encoded and unfolded during transport with hsp60 chaperone proteins, targeted by a signal sequence for importation into organelle

Sorting Proteins to Mitochondria & Chloroplast

Have their own genomes and ribosomes, but most proteins for these organelles are nuclear encoded

Mitochondria & Chloroplast Sorting

Co-translational sorting, proteins are nuclear encoded and have an ER signal sequence, associated with ER during protein synthesis in cytosol, ER signal sequence is hydrophobic

Sorting Proteins to ER

Entry point to ER, golgi apparatus, endosomes, lysosomes

Why do Proteins sort to ER

mRNA arrives in cytoplasm, translation starts on ribosomes in cytosol, translating a protein with ER sequence, insertion of protein into ER starts as translation continues, proteins entering ER: soluble proteins, transmembrane proteins

Steps of Sorting into ER

1. Translation starts, N-terminal ER signal sequence emerges
2. Recognized by SRP, elongation arrest by SRP
3. SRP ribosome complex -\> SRP receptor -\> Protein Translocator
4. Protein Translocator Opens
5. Protein synthesis resumes with protein transfer into ER lumen
6. Signal peptidase cleaves signal sequence (signal sequence is hydrophobic, in lipid bilayer)
7. Protein released into ER lumen
8. Protein translocator closes
Destination of soluble protein: lumen of endomembrane organelle or secretion at plasma membrane

Co-Translational Translocation: Soluble Protein

Signal recognition particle, recognizes signal sequences

SRP

1. Translation starts, N-terminal ER signal sequence emerges
2. Recognized by SRP, elongation arrest by SRP
3. SRP ribosome complex -\> SRP receptor -\> Protein Translocator
4. Protein Translocator Opens
5. Protein synthesis resumes with protein transfer into ER lumen
6. Stop-transfer sequence enters Translocon (internal hydrophobic segment \= membrane spanning alpha helix)
7. Protein transfer stops and transmembrane domain released into lipid bilayer
8. Signal peptidase cleaves ER signal sequence and translocon closes
9. Protein synthesis completed

Co-Translational Translocation: Transmembrane Protein

At N-terminus of protein: stretch of hydrophobic amino acids, removed by signal peptidase

N-Terminal ER Signal Sequence

Inside of the protein, start transfer sequence, stretch of hydrophobic amino acids, not removed remains a part of the protein \= membrane spanning alpha helix

Internal ER Signal Sequence

1. Internal start transfer sequence emerges
2. Recognized by SRP, elongation arrest by SRP
3. SRP ribosome complex -\> SRP receptor -\> Protein Translocator
4. Protein Translocator Opens
5. Protein synthesis resumes with protein transfer into ER lumen
6. Stop transfer sequence enters translocon
7. Protein transfer stops
8. Start-transfer sequence and stop-transfer sequences are released into lipid bilayer, hydrophobic segments \= membrane spanning alpha helices
9. Translocon closes
10. Protein synthesis completed
Destination: Membrane of endomembrane organelle or in plasma membrane

Co-Translational Translocation: Transmembrane Protein with Internal Sequence

Orientation of hydrophobic segment depends on flanking positively charged amino acids, plus end is oriented in cytosol

Internal Start Transfer Sequence

Endoplasmic Reticulum, Golgi Apparatus, Endosomes, Lysosomes form endomembrane system, intracellular compartments exchange lipids and proteins

Endomembrane System

Proteins and lipids made in ER delivered to other compartments
ER to outside (exocytosis)
ER to lysosomes via endosomes

Secretory Pathway

Contents move into cell

Endocytic Pathway

Retrieval of lipids, selected proteins for reuse

Retrieval Pathway

Vesicle contents delivered to extracellular space, vesicle membrane becomes part of plasma membrane

Exocytosis

Vesicle luminal contents come from extracellular space, plasma membrane forms vesicle membrane, contents brought into cell

Endocytosis

Small membrane enclosed organelle in cytoplasm of a eukaryotic cell, shuttles components back and forth in the endomembrane system, for example from ER to golgi, cargo receptors can select cargo

Vesicle

Is in all eukaryotic cells, continual delivery of transmembrane and soluble proteins and lipids to plasma membrane, includes constitutive secretion of proteins for example collagen or ECM

Constitutive Exocytosis Pathway

regulated secretion in specialized cells, stored in specialized secretory vesicles, extracellular signal leads to vesicle fusion with plasma membrane and contents released, for example pancreatic beta cells with insulin release during increase blood glucose

Regulated Exocytosis Pathway

1.Translation starts on cytosolic ribosomes: ER signal at N-terminus directs protein to ER
2. Co-translational translocation at ER: protein inserted through ER membrane by translocon protein, ER signal sequence cleaved and left behind in ER membrane, secreted protein ends up in ER Lumen
3. Secreted protein: Moves in transport vesicles, through secretory pathway, ER to golgi apparatus to plasma membrane, vesicle membrane fuses with plasma membrane during exocytosis, secreted protein released to extracellular space

Path of a Secreted Protein

Each membrane protein has a specific orientation, is a result of membrane insertion into ER, this protein asymmetry is maintained through vesicular transport

Maintenance of Membrane Protein Asymmetry

Receives proteins and lipids from the ER and modifies them, and then dispatches them to other destinations in the cell

Golgi Apparatus

Protein glycosylation starts in the ER, single type of oligosaccharide is attached to many proteins, complex oligosaccharide processing occurs in golgi apparatus, a multistage processing unit, different enzymes in each cisterna, glycosylation modifications for lipids and proteins

Protein Glycosylation

Membrane bound organelles, material ingested by endocytosis is transported to early endosomes and sorted, early endosomes mature into late endosomes

Endosomes

Membrane bound organelles, contain hydrolytic enzymes to digest worn out proteins and organelles and other waste, late endosomes mature into lysosomes, lysosomal hydrolase is transported from ER to golgi to endosomes

Lysosomes

Lysosomes: contain 40 types of hydrolytic enzymes: acid hydrolases, proteases, nucleases, lipases. Lysosomes are acidified by H+ pump, low pH needed for hydrolytic enzymes

Main Site of Intracellular Digestion

On the noncytosolic face, glycosylated for protection from proteases, organelle membrane protects rest of cell from digestion

Lysosomal Membrane Proteins

Transfer digested products to cytosol, for example amino acids, sugars, nucleotides

Transport proteins in lysosomal membrane

1. Translation starts on cytosolic ribosomes, ER signal sequence (N-terminal or internal), directs protein to ER
2. Co-translational translocation at ER, protein inserted into ER membrane by a translocon protein
3. Transmembrane protein: moves in transport vesicles through secretory pathway, ER to golgi apparatus to plasma membrane, vesicle fuses with plasma membrane during exocytosis, transmembrane protein transferred to plasma membrane

Path of a Transmembrane Protein

Specialized material outside of the cell

Extracellular Matrix

Cell wall, vacuoles, chloroplast

Plant Organelles not found in Animal Cells

Cell shape, protection against mechanical stress

Cell Wall

2 Types: Degradation and storage of small molecules and proteins

Vacuoles

Site of photosynthesis

Chloroplast

Contents of the cell outside of the nucleus

Cytoplasm

Aqueous part of the cytoplasm

Cytosol

Inside of organelles

Lumen

Compartmentalization, Scaffold for biochemical activities, selectively permeable barrier, transport solutes, respond to external signals, interactions between cells

Cellular Functions at Membranes

Two layers or leaflets of lipid molecules form a membrane, composed of many different lipids: phospholipids, sterols, glycolipids

Membrane Bilayers

Must have glycerol group \= phosphoglycerides, different types of phosphoglycerides result from differences in hydrophilic polar head group, length is about 14-24 carbon atoms, saturated or unsaturated, have hydrophobic tail

Phospholipids

Result of an unsaturated tail, contains a cis-double bond, increase fluidity at low temperatures as they reduce packing

Kink in Phospholipids

Spontaneously self associate into a bilayer, polar head group interacts with water, two hydrophobic hydrocarbon tails interact with other hydrophobic tails

Phospholipids in Aqueous Environment

Artificial bilayers, used to study lipid properties, membrane protein properties, drug delivery into cells

Liposomes

Laser tweezers are used to manipulate membrane since membrane can be deformed without causing damage

Live Cell Imaging

Phospholipids within each leaflet rapidly diffuse laterally, rotate, flex, but rarely move from one leaflet to other (flip-flop) on their own

Phospholipid Movement

Carefully regulated as important for function, e.g. membrane proteins - transport, enzyme activity, signaling

Cell Membrane Fluidity

Lower temperatures cause more viscous less fluid membrane, cis-double bonds increase fluidity at lower temperatures as they reduce tight packing, shorter hydrocarbon tails increase fluidity at lower temperatures as lipid tails interact less, addition of cholesterol stiffens membrane and is less permeable to water

Factors Affecting Membrane Fluidity

(Sterols in plants mainly) Decrease mobility of phospholipid tails, stiffens membrane, plasma membrane is less permeable to polar molecules

Cholesterol

Enzymes in the ER or Golgi membrane flip lipids from one leaflet to the other

Lipid Movement to the Other Leaflet

Phospholipid Translocator, catalyzes rapid flip flop of random phospholipids from one leaflet to the other, needed as phospholipids are synthesized in the cytosolic leaflet of ER

Scramblase

Membranes retain orientation of cytosolic/noncytosolic face

Asymmetry of Lipid Bilayer

Catalyzes rapid flip flop of specific phospholipids to the cytosolic leaflet, e.g. phosphatidylserine, some can bind cytosolic proteins at plasma mebrane, e.g. phosphatidyl serine binds protein kinase C

Flippase

Formed by adding sugar groups to lipids/proteins on luminal face of Golgi, end up inside of some organelles on the noncytosolic face, protect membrane from harsh environments

Glycolipids and Glycoproteins

Proteins directly attached to lipid bilayer, inserted into bilayer or attached to a lipid inserted into bilayer

Integral Membrane Proteins

Use of detergent which destroys lipid bilayer

Extraction Method of Integral Membrane Proteins

Proteins do not insert into membrane, on either face, bound to other proteins or lipids by non covalent interactions

Peripheral Membrane Proteins

Gentle extraction methods used, lipid bilayer intact

Extraction Method of Peripheral Membrane Proteins

Amphipathic, have hydrophilic domains: aqueous amino acid side chains are polar, hydrophobic membrane spanning domains: amino acid side chains are nonpolar, can be a single helix \= single pass, multiple helices \= multipass, or barrel of beta sheets

Transmembrane Proteins

Each transmembrane protein has a specific orientation which is essential for function, types include transporters and channels, anchors, receptors, enzymes

Functions of Transmembrane Proteins

X-ray crystallography determines 3D structure, hydrophobicity plots show segments of 20-30 hydrophobic amino acids that span the lipid bilayer

How Transmembrane structures identified?

Proteins anchored on cytosolic face by an amphipathic alpha helix, for example proteins in membrane bending Sar1, found on cytosolic side

Monolayer-Associated Membrane Proteins

Lipid linked membrane protein, synthesis in ER lumen, end up on cell surface (noncytosolic face)

Proteins with GPI Anchor

Cytosolic enzymes add anchor, directs protein to cytosolic face

Protein with Fatty Acid or Prenyl Anchor

Done through Fluorescence Recovery after photobleaching, protein fused to green fluorescent protein, area is photobleached, recovery: neighboring unbleached fluorescent proteins randomly move around, migrate in

Study of protein Movement

Time taken for neighboring unbleached fluorescent proteins to move into bleached area

Rate of Fluorescence Recovery

Impermeable to most water soluble molecules

Permeability of Liposome

Membrane transport proteins to transfer specific molecules called facilitated transport

Permeability of Cell Membrane

Small nonpolar and uncharged molecules move through simple diffusion high concentration to low concentration more hydrophobic or nonpolar molecules results in faster diffusion, ions and larger uncharged polar molecules require membrane proteins to transport

Movement across the Lipid Bilayer

Transmembrane transport proteins create a protein lined path across cell membrane that transport polar and charged molecules, each transports a specific class of molecules, different cell membranes have a different complement of transport proteins

Proteins involved in mebrane transport

Selects cargo based on size and electrical charge of solute, transient interactions as solute passes through, no conformational changes for transport through an open channel

Channel Membrane Transport Protein

Selects cargo based on how solute fits into binding site, specific binding of solute, series of conformational changes for transport

Transporter Membrane Transport Protein

Requires an input of energy to transport

Active Transport

Does not require additional energy and occurs naturally

Passive Transport

Concentration gradient + Membrane potential

Electrochemical gradient