Chapter 15 : Intracellular Compartments and Transport

Outline of Chapter Content

1. Membrane-Enclosed Organelles

  • Help achieved cellular compartmentalization

  • Primary functions of the membrane-enclosed compartments of eukaryotic cell :

Compartment Main Function

Cytosol Metabolic pathways, protein synthesis

Nucleus Contain main genome, DNA/RNA synthesis

ER Synthesis of most lipids, protein distribution

Golgi Protein and lipid modification for distribution

Lysosomes Intracellular degradation

Endosomes Sorting of endocytosed materials

Mitochondria ATP synthesis- oxidative phosphorylation

Chloroplasts Photosynthesis

Peroximes Oxidative of toxic compounds

2. Protein Sorting Mechanisms

  • Energy-dependent Mechanisms for protein entry into an organelle from the cytosol

  • There are three mechanisms :

  1. Transport Through Nuclear pores ( Proteins remain folded)

  • Nucleus composed of two membranes :

° Inner membrane

° Outer membrane ( continuous with the ER membrane )

  • Nuclear pores are gates on both membranes that allow movement in both directions.

  • Each is a complex of approximately 30 different proteins; total of approximately 450 proteins molecules

  • Small molecules can easily diffuse.

  • Large molecules like proteins need an appropriate sorting signal ( called nuclear localization signal; short stretch of lysines/arginines, positives charges) and helper proteins ( called nuclear transport receptors ).

  • Proteins transport through nuclear pores required energy ( active ) that is provided by the hydrolysis of GTP, mediated by a GTPase named Ran ( Ran-GTP nucleus & Ran-GDP cytosol)

° Ran-GAP ( GTPase-activating protein), is found exclusively in the cytosol

° Ran-GEF ( guanine nucleotide exchange factor), is found exclusively in the nucleus `

  • Ran-GEF ( in nucleus) converts Ran-GDP to Ran-GTP, creating high Ran-GTP levels inside the nucleus.

  • Ran-GAP ( in cytosol) converts Ran-GTP to Ran -GDP, creating high Ran-GDP levels in the cytosol.

  • This gradient ensures that import receptors release their cargo inside the nucleus when they bind Ran-GTP and then return to the cytosol carrying Ran-GTP.

  • In the cytosol, Ran-GTP is hydrolyzed to Ran-GDP, releasing the receptor so it can bind a new cargo.

  1. Transport across mitochondrial and chloroplast membranes (Proteins have to be unfolded)

  • Mitochondrial proteins are made in the cytosol and contain an N-terminal signal sequence ( positively charged amphipathic helix) that directs them to the mitochondrion.

  • The signal sequence binds specific import receptors on the outer membrane, and the protein is threaded through both membranes via aligned translocators ( TOM on the outer membrane TIM on the inner membrane).

  • The protein must be unfolded to cross the membranes, and once inside the matrix, chaperonins refold the protein and the signal peptide is cut, producing the mature mitondrial protein.

  1. Transport Across the ER membrane (Proteins remain folded )

  • ER most extensive membrane system in the cell

  • Entry point for proteins for destined for several other organelles and the cell exterior.

  • Two kinds of proteins are transferred from the cytosol to ER :

  1. Soluble proteins

  • Transferred completely to the ER lumen

  • Destined for secretion out of the cell or the or to the lumen of another organelle ( not mitochondrion or plastid )

  1. Transmembrane proteins

  • Partly translocated into the ER, embedded in membrane.

  • Destined to reside in the ER or the membrane of another organelle or plasma membrane

  • Both are initially directed to the ER by an ER signal sequence ( N-terminal stretch of hydrophobic amino acids)

  • Retention in the lumen of the ER requires a signal sequence at C-terminus ( KDEL)

Proteins Enter the ER while being synthesized

  • Proteins destined for the ER contain an N-terminal signal sequence that directs the ribosome to the ER membrane during translation

  • When the signal sequence appears, SRP ( signal recognition particle ) binds it and pauses translation

  • SRP carries the ribosome having the nascent polypeptide to the SRP receptor on the ER membrane.

  • SRP is released and recycles, while the ribosome is transferred to a protein translocation channel.

  • Translation resumes, and the growing polypeptide is inserted into the ER lumen as it is synthesized.

Transport of soluble proteins into the lumen of the ER

  • Signal sequence remains bound to the translocation channel while the rest of the protein is threaded through as a loop.

  • Once completed, the signal peptide is cleaved ( cut).

Integration of a transmembrane protein into the ER membrane

  • A start-transfer sequence ( hydrophobic) initiates insertion by opening the translocation channel and letting part of the protein enter the lumen.

  • A stop-transfer sequence ( hydrophobic) halts translocation and causes that region of the protein to exit sideways into the membrane, becoming the transmembrane domain.

  • The signal peptide ( if present) may be cleaved by signal peptidase, but the stop-transfer sequence remains as the anchored membrane segment.

  • Final protein orientation ( which sides faces the cytosol vs lumen) is set during insertion and is maintained through all vesicle trafficking to the Golgi, lysosomes, or plasma membrane.

Integration of multi-pass transmembrane protein

  • Here, an internal start-transfer sequence is used and the protein is not cleaved.

Signal Sequences in Protein Sorting

  • They act as “address labels” that direct proteins to the correct organelle.

  • Necessary because without the signal, a protein will stay in the cytosol.

  • Sufficient because adding the signal to any protein is enough to redirect it into the ER.

  • For many signals, the structural features ( like being hydrophobic) matter more that the exact amino-acid sequence.

2. Vesicular Transport

  1. Vesicle Transport Inside the cell

  • ER to Golgi ( forward/ anterograde transport)

  • Golgi to ER ( backward/ retrograde transport)

  1. Exocytosis ( Secretory Pathways )

  • Vesicles from the Golgi fuse with the plasma membrane

  • This releases proteins outside the cell

  • Also inserts new membrane proteins and lipids into the cell surface

  • Protein modification in the ER

  • Controlled exit from ER

  • Protein modification and sorting in the Golgi

  1. Endocytosis ( Endocytic Pathways )

  • Materials at the plasma membrane are engulfed into early endosomes

  • Later mature into late endosomes

  • Late endosomes fuse with lysosomes for degradation.

  1. All of these pathways = The Endomembrane System

It includes

  • ER

  • Golgi

  • Transport vesicles

  • Plasma membrane

  • Endosomes

  • Lysosomes

Everything is connected through vesicles budding and fusion.

Specificity is required for the cargo and the destination

  • Vesicles must pick the correct cargo and deliver it to the correct destination compartment.

  • Specificity depends on the vesicle membrane and proteins and proteins on the target membrane.

  • Recognition ensures

  1. The right proteins enter the vesicle

  2. The vesicle fuses only with the proper organelle

How Vesicles Select the correct cargo

  • The cytosolic side of membranes contains coat proteins that help select cargo.

  • Coat proteins shape the membrane into a bud and helps capture molecules.

  • Transport receptors recognize specific cargo proteins

  • Adaptins ( e.g : Clathrin- coated —- clathrin +adaptin 2—- plasma membrane to endosomes) trap transport receptors and attach them to clathrin

Note that :

  • Plasma membrane - specific adaptins

  • Golgi- specific adaptins

Vesicular Transport - Specificity for the target membrane

  • A vesicle must fuse only with the correct target membrane

  • Incorrect fusion would mix organelle contents and disrupt cell function.

  • Specificity comes from molecular markers on both the vesicle and target membranes called SNARES ( related transmembrane proteins).

Types of Snares

  • V-SNARES — on the vesicle

  • t-SNARES — on the target membrane

  • v-SNAREs bind only to their matching t-SNAREs

  • This ensures vesicles dock and fuse only with the correct membrane

  • The SNARE pairing pulls the two membrane close, driving membrane fusion.

Rab Proteins and Tethering Proteins

  1. Rab proteins

  • Small GTP-binding proteins located on vesicles.

  • Each Rab type directs a vesicle to a specific target membrane.

  1. Tethering proteins

  • Located on the target membrane.

  • Recognize and bind to the correct Rab, providing the first level of specificity.

  • This step is called tethering.

Steps in Vesicle Targeting and Fusion

  • Vesicle forms with Specific cargo and v-SNARE + Rab on its surface.

  • Rab binds to a matching tethering protein on the target membrane

  • v-SNARE binds to matching t-SNARE — docking

  • SNARE complex “sippers” together and brings membranes extremely close.

  • Fusion occurs —- cargo delivered into the target organelle or membrane

  • SNAREs are later separated and recycled.

3. Protein modification in the ER

3.1 Disulfide bond formation : oxidation ( e.g insuline) of pairs of cysteine side chains ( cystine) and stabilizes protein structure.

  • Interchain disulfide bond = a covalent bond formed between the sulfur atoms of cysteine residues from different polypeptide chains, which plays a crucial role in stabilizing the tertiary and quaternary structures of proteins.

  • Intrachain disulfide bonds are formed between cysteine residues within the same polypeptide chain, contributing to the overall stability and correct folding of the protein.

3.2 Glycosylation : addition of pre-assembled 14-sugar oligosaccharide ( glucose, manose, N-acetylglucosamine) to proteins inside the ER.

  • Added to the amide nitrogen of asparagine ( Asn) — N-linked glycosylation ( via Oligosaccharyl transferase )

  • Occurs inside the ER lumen as the protein is being translocated

  • Helps protein folding by signaling to chaperones.

  • Protects proteins from degradation.

  • Acts as a sorting signal for protein trafficking.

Exit of proteins from the ER is controlled

  • Chaperonins prevent misfolded proteins or unassembled complexes from leaving the ER.

  • Misfolded proteins bound to sensors that activate the unfolded protein response (UPR), which leads to an increase in chaperone production.

Protein modification and sorting in the Golgi

  • The Golgi consists of multiple stacks of flattened membrane sacs called cisternae.

  • Each stack has two faces

Cis-Golgi — faces the ER ( receives vesicles from the ER)

Trans-Golgi — faces the plasma membrane ( ships vesicles out)

Protein Processing in the Golgi

  • Proteins arrive at the cis-Golgi from the ER in vesicles

  • They move through cisternae via vesicular transport or cisternal maturation

  • The Golgi performs additional modifications, including :

  1. Further glycosylation ( trimming and adding sugars )

  2. Modification of oligosaccharides ( mannose— complex sugars)

  3. Addition of localization signals for proper sorting

Protein sorting in the Golgi

  • The trans-Golgi functions as the main sorting station.

  • Proteins are directed to different destinations based on sorting signals :

Plasma membrane

Lysosomes

Secretory vesicles

Back to the ER ( via KDEL sequence)

5. Exocytosis & Endocytosis

5.1 Exocytosis

  • Constitutive:

  1. Continuous operation

  2. Supplies new lipid and protein to the plasma membrane

  3. Secretion of proteins to outside of cell

  4. Default pathway

  • Regulated :

  1. Operates only in specialized secretion cells

  2. Secretory vesicles concentrate at plasma membrane but will not fuse until triggered by an appropriate signal

  3. Vesicles contain surface signals and distinct ionic conditions that proteins to aggregate

  • Both diverge in the trans Golgi network

5.2 Endocytic Pathways - Endocytosis

  • Inward movement of fluid, molecules and even cells

  • Endocyotic vesicles formed from plasma membrane

Phagocytosis ( cellular eating ): Ingestion of large molecules or cells via large vesicles; specialized phagocytic cells

  • Common mechanism for feeding in protozoa

  • Important in animals for defense against infection and for recycling dead/damaged cells.

Pinocytosis ( cellular drinking ) : Ingestion of fluid and small molecules via small vesicles; All eukaryotic cells, all of the time.

  • Endocytosis via clathrin-coated vesicles

  • After shedding the clathrin coat, the vesicles fuse with the endosome, releasing the extracelluar fluid and dissolved substances.

Indiscriminate pynocytosis

  • Fluid and dissolved molecules simply trapped

  • Renewal of plasma membrane

  • ALL cells, all the time

Receptor-mediated endocytosis

  • isUptake of specific molecules that bind to complementary receptors on the cell surface.

  • Allow selective concentration of molecules

  • E.g : Cholesterol uptake

  • Cholesterol is required for membrane synthesis and other essential functions

  • In the bloodstream, cholesterol is carried inside low-density lipoproteins ( LDL)

Steps of LDL Uptake

  • LDL binds to LDL receptors on the plasma membrane

  • The receptor-LDL is internalized via receptor-mediated endocytosis ( Clathrin-coated vesicles)

  • Vesicles fuse with the early endosome

Role of Endosomes

  • Early endosomes are acidic, causing LDL to dissociate from its receptor

  • LDL receptor are recycled back to the plasma membrane via transport vesicles.

  • LDL is trafficked from the endosome to the lysosome.

Lysosomal processing

  • in the lysosome, LDL is broken down by enzymes.

  • Free cholesterol is released into the cytosol for use by the cell.

5.3 The lysosome : Intracellular digestion

  • Lysosomes are acidic organelles containing M6P-targeted hydrolases that digest macromolecules and export the resulting nutrients back to the cytosol.

  • Contain acid hydrolases ( nucleases,proteases,lipases,etc..) that digest wide variety of biological molecules.

  • These enzymes only function at low pH ( approx 5), maintained by an ATP-driven H+ pump.

  • The lysosomal membrane contains transported to move digestion products ( amino acids, sugars, nucleoides) back to the ciytosol.

How Lysosomal Enzymes are targeted

  • They are modified in the cis-Golgi by addition of a mannose-6-phosphate (M6P) tag.

  • The M6P signal directs these enzymes to vesicles that deliver them to the lysosome.

Somes of the approx 50 lysosomal storage diseases

  • Tay-Sachs

  • Batten’s disease

  • Niemann-Pick

  • Gaucher disease

  • Mucopolysaccharideosis

  • Leukodystrophy

Collective incidence : 1/7700 births