Cell Biology Chapter 11: Protein Sorting and Transport 낱말 카드 | Quizlet

Endomembrane System

Network of communicating membrane with related functions in the cell

-Communicate via vesicular transport

-Function in protein processing and lipid synthesis

Where does protein synthesis take place?

On ribosomes in the cytoplasm (i.e. either free in the cytosol of on the RER)

Where does protein synthesis begin?

On ribosomes that are free in the cytosol

Are proteins ready to function once synthesized?

No; proteins need to fold and typically go through a variety of processing events as well

Events "After" Protein Synthesis

Folding

Protein cleavage

Glycosylation

Lipid attachment

Protein phosphorylation

Protein Targeting

Bold=Protein Modification

Protein Targeting

Involves:

Specific signal sequence

Receptors & recognition protein

Chaperones (in some cases)

Endoplasmic Reticulum (ER)

Network of membrane enclosed

Two sides:

Lumenal (lumen) vs. Cytosolic (cisternal space)

Smooth ER

No ribosomes

Lipid Metabolism

Tubules

Rough ER

Ribosomes

Protein synthesis and processing

Flat sacs

Protein Sorting

Some proteins can synthesis free in the cytosol or on membrane bound ribosomes and release into either in the cytosol or ER lumen where they are later taken to an organelles to fulfill its function.

Secretory Pathway

RER > Golgi > Secretory Vesicle > Cell Exterior

Pulse Chase Experiment

Studies the pathway of secreted proteins by labeling new synthesized proteins with radioactive amino acids

1. Pancreatic cells were exposed to AA, then were found in the RER

2. Incubated with nonradioactive AA the cells were found in the Golgi Apparatus

3. After longer chase period the cells went from the Golgi Apparatus to secretory vesicles.

4. Secretory vesicles fuse with plasma membrane to be released outside the cell

Protein Targeting

How protein are targeted to ER

Signal Sequence

At AA N-terminus of the polypeptide chain (polymer of AA)

Short stretch of hydrophobic AA that are cleaved from the polypeptide chain during its transfer into the ER lumen

Span about 20 AA

Cotranslational Targeting

1. As the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP)

2. The SRP escorts the complex to the ER membrane where it binds to the SRP receptor

3. The SRP, is released, the ribosome binds to the translocon and insertion of the signal sequence opens the translocon.

4.Translation resumes and the signal sequence is cleaved by signal peptidase

5. Continued translation drive the translocation of the growing polypeptide chain across the membrane.

6. The complete polypeptide chain is released within the ER lumen.

Signal Recognition Particle (SRP)

Composed of:

6 polypeptide

srpRNA (a 7s RNA)

Post-Translational Targeting

NO SRP

1. Synthesized on free

ribsomes

2. Cytosolic chaperones maintain unfolded conformation

3. Recognized by the Sec 62/63 complex and SS in inserted into the translocon in the ER membrane.

4. The Sec 63 protein is associated with a chaperone protein called BiP.

5. The binding of multiple BiP molecules and the their release gives off ATP hydrolysis which drives the translocation of the polypeptide chain into the ER lumen.

Stop Transfer Sequence

Terminal signal sequence cleaved

Internal Signal Sequence

Signal sequence is not cleaved

Processing and other events in the ER

Protein folding

-chaperones: catalyze protein folding (e.g. BiP, hsp's, calnexin, calreticulin)

-Assembly of multisubunit proteins

-Disulfide bond formation

protein disulfide isomerase

-Initial stages of glycosylation

-Glycolipid anchors

Chaperones

Essential in many processes in cell

Increase the rate of processes related to protein structure, conformation, folding

Lipid Synthesis

Happens in the SER

Membrane lipids such as phospholipids are synthesized by using:

-on pre-existing membranes

-using water-soluble cytosolic precursors (hydrophobic)

Lipids get to other membranes in the cell by: Transporting in the vesicles or by carrier proteins

Phospholipid Synthesis

1. Glycerol-3-Phosphate is synthesized on the cytosolic side of ER membrane from water soluble precursors.

2. Fatty acids are first transferred from coenzyme A carrier to glycerol-3-phosphate by membrane-bound enzymes, and the result of phosphatidic in inserted into the membrane.

3. A phosphate then converts phosphatidic acid to diacylglycerol

4. Catalyzation of a additional different polar heads occur via changing of glycerol by enzymes.

Bilayer

Since the membrane is a bilayer more phospholipids are produced on one side than the other

Flippases

By catalyzing the rapid translocation of phospholipids across the ER membrane, the flippases ensure even growth of both halves of the bilayer

Endomembrane System: Regulation of Transport

Nature of signals:

Peptide sequence

-short stretch of AAs in linear sequence (e.g. KDEL)

Signal patch or Conformation

-series of AA's brought together by 3D strc vs. linear sequence

Carbohydrate

Types of signals:

Export

Retention/Retrieval

Default

ER Export Signal

Transmembrane proteins:

-Di-acidic or di-hydrophobic signals bind to cytosolic adaptor proteins

-Also act as receptors for lumenal and GPI-anchored proteins

Lumenal proteins:

-Signal patch, conformation or default pathway

GPI anchors:

-Recognized by the GPI anchors

Di-acidic = Asp-Asp or Glu-Glu

Di-hydrophobic = Met-Met

Asp = aspartic acid

Glu = glutamic acid

Met = methionine

GPI=glycosylphosphatidylinositol

ER Retention/Retrieval Signals

-Also called retrograde transport

-Targets to specific location through specific recycling pathway

-Signal recognized by recycling receptor

-Some proteins retrieved by binding to proteins w/

such signals

KDEL

Soluble in ER

K=lysine D=aspartic acid E=glutamic acid L=leucine

KKXX

Transmembrane ER

Default Signals

Lack of signal?

Bulk flow through secretory pathway?

Unsure if may be an active signal

Secreted or cell surface

Polarized Cells

Plasma membrane has two domains:

Apical

Basolateral

Signals direct to membrane proteins to one membrane surface or other

Variety of signals involved

Golgi

Golgi Apparatus

Golgi Complex

What is the Golgi function?

Further processing of protein and sorting them to their final destination

Targets to: intracellular destinations

Cell Surface

-via recycling endosome

-Constitutive pathway

-Regulated secretory pathway (stored in secretory vesicles)

What is more of the Golgi functions?

Synthesis of lipids

-Glycolipids & sphingomyelin

-Ceramide precursor synthesized in ER

Plants: complex cell wall polysaccharide

-Cellulos, straight chain, synthesis at cell surface by plasma membrane enzyme

-Hemicelluloses & pectins, branched chain, synthesized in Golgi

Structure of the Golgi

Series of separate flattened membrane-enclosed sacs (cisternae)

Distinct polarity in both structure and function

Structural Polarity:

Cis Face

-entry of molecules from ER, usually side closer to nucleus

Trans Face

- Exit golgi on route to final destination, usually side closer to cell surface

Polarity Function in Golgi

Proteins move within compartments that mature:

-Cisternal migration/progression

-Golgi components return to previous sections by retrograde flow within vesicles

Compartments:

-ER-golgi IC

-Cis golgi network

-Trans golgi network

Orientation of Membrane Surface

Topologically lumen is equivalent to cell exterior

What is glycosylation?

Modification of the carbohydrate portion of glycolipids

What is protein glycosylation?

It is the addition of a carbohydrate to protein by a covalently attached of an amino acid side chain

What are the two types of protein glycosylation?

N-linked and O-linked

N-linked

-CH2On attached to N of asparagine

-Begun in the ER further processed in Golgi

O-linked

-CH2On attached to O of Serine or Threonine

-Occur in Golgi

N-linked vs O-linked

-CH2On attachment point

-Location where addition begins

-Mechanism: addition and modification of performed structure vs sequential addition

What determines glycosylation pattern of a protein?

Structure of protein

-Appropriate AA and signal

-Accessibility of region

Amount processing enzymes present with in Golgi of different types of cells

Carbohydrates modification is important in targeting of proteins to lysosomes because the chain carries information

N-linked glycosylation (1)

Occurs in golgi

The N-linked oligosaccharides of glycoproteins transported from the ER and are further modified by an ordered sequence of reaction catalyzed by enzymes in different compartments of the Golgi.

N-linked glycosylation (2)

For lysosomal targeting occurs in the cis golgi

Proteins destined for incorporation into lysosomes are specifically recognized and modified by the addition of phosphate groups to the number 6 position of mannose residues. In the first step of the reaction, N-acetylglucosmine phosphates are transferred to mannose residues from UDP-N-acetylglucosamine group is then removed, leaving mannose-6-phosphate.

Vesicular Transport

Targeting

Establishes and maintains the functional organization of the cell

Selectivity of transport

-is the key to appropriate targeting

-determined by combination of vesicle coat proteins, GTP binding proteins and other associated proteins

Vesical coat proteins

are involved in specific trafficking routes

What are coat proteins and GTP-binding proteins?

Vesicle Coat Proteins:

Clathrin

COP I

COP II

Small GTP-binding proteins:

ARF 1-3

Snar 1

Rab protein family

Formation of Transport Vesicle

Activities:

-sorting of cargo

-budding of vesicle

Players:

-Coat proteins=Clathrin

-Adaptor proteins=GGA, AP1

-GTP-binding protein=ARF1

Clathrin

-Structural Role

-Basket-like lattice

-Distorts membrane

Adaptor Proteins

-Medites clathrin binding

-Selects vesicle contents

Transport of lysosomal proteins by clathrin-coated vesicles

-Lysosomal protein bound on lumenal face to R

-R=transmembrane proteins

-Adaptors (GGA & AP1) binds cytosolic portion of R

-Designates clathrin... lysosomal target

GTP-binding protein

ARF1 initiates vesicle budding

-ARF is activated by ARF-GEF: ARF(GDP) > ARF(GTP)

-ARF (GTP) recuits adaptor proteins: GGA

-GGA recruits receptor which carries the lysosomal protein (a lysosomal hydrolase)

-GGA also recruits AP1 which is a binding site for clathrin

Movement of Transport Vesicle

Activities

-Recycle ARF, coat proteins and adapters

-Travel along cytoskeleton

Players

-GTP hydrolysis on ARF weakens coat

-Hsp 70s remove some of coat proteins

-Remaining coat proteins bind tubular and molecular motors

Membrane Fusion

involves protein-protein interactions, the assembly of protein complexes, and GTP hydrolysis

Lysosomes

-Membrane enclosed organelle

-Vary size & shape

-Enzyme digest all biological polymers

Functions of Lysosome

1. Digest material taken up by endocytosis

2. Phagocytosis

-Cleaning up debris via macrophages

3. Autophagy

-Programmed cell death

-The turnover of the cell's own components

Organization of Lysosome

-Acid hydrolyses (ph5)

-Expend energy maintain internal pH.

The acidic internal pH of lysosomes results from the action of a proton pump in the lysosomal membrane, which imports protons from cytosol coupled to ATP hydrolysis.