unit 3 cell and molec

Nucleus

Nucleus: site of DNA and RNA synthesis

Ribosomes

Ribosomes: site of protein synthesis

Cytoplasm

Cytoplasm: intermediary metabolism and protein synthesis (translation); consists of cytosol and organelles

Endoplasmic Reticulum

Endoplasmic reticulum: smooth ER and rough ER

Golgi Apparatus

Golgi apparatus: protein modification and sorting

Endosomes

Endosomes: sorting and recycling of endocytosed material before reaching lysosome

Lysosomes

Lysosomes: intracellular digestion

Mitochondria

Mitochondria: aerobic respiration

Peroxisomes

Peroxisomes: oxidative reactions and breakdown of long-chain fatty acids

Protein Sorting: Signal Sequence-Mediated Targeting

Proteins contain short amino acid “tags” (signal sequences) that direct them to specific organelles; cellular machinery recognizes and transports accordingly

Signal Sequence Example

Nuclear localization signal (NLS) directs proteins to the nucleus

Protein Sorting: Vesicular Transport

Proteins are packaged into vesicles that bud from one compartment (ER) and fuse with another (Golgi)

Vesicular Transport Example

Secretory proteins move ER → Golgi → plasma membrane

Protein Sorting: Retention/Retrieval Signals

Proteins contain motifs that ensure they stay in or are retrieved back to a compartment if they escape

Retention Signal Example

KDEL sequence ensures ER retention/retrieval

Topologically Equivalent Compartments

Compartments are topologically equivalent if molecules can move between them without crossing a membrane

type of sorting signals:

Signal Sequences (Linear)

Short continuous amino acid sequences that direct proteins to organelles (e.g., ER signal peptide)

types of sorting signals: Signal Patches (3D)

Non-contiguous amino acids that form a targeting signal after protein folding (e.g., mitochondrial targeting signals)

Signal Sequence vs Patch

Linear sequence = signal sequence; folded motif = signal patch

Cell Compartments Overview

Eukaryotic cells contain membrane-bound organelles with specialized functions

Importance of Protein Sorting

Each organelle requires correct proteins; proteins are made in cytosol or ER and targeted via signals, vesicles, or retention motifs

Organelle Biogenesis

Organelles arise from pre-existing organelles, not de novo

Nuclear Envelope Structure

Nuclear envelope consists of two membranes: inner and outer nuclear membranes

Inner Nuclear Membrane

Lines nucleus; provides structural support; interacts with chromatin and nuclear lamina

Outer Nuclear Membrane

Continuous with rough ER; may have ribosomes attached

Nuclear Pores

Protein complexes spanning both membranes allowing selective transport

Perinuclear Space

Space between membranes; continuous with ER lumen

Nuclear Pore Complex Function

Regulates bidirectional movement of proteins and RNA; maintains compartmentalization

NPC Capacity

Each NPC can transport up to 1000 macromolecules/second; cells contain ~3000–4000 NPCs

Passive Transport (NPC)

Small molecules diffuse freely; no energy required; limited by size

Passive Limit

Proteins >60,000 daltons cannot passively diffuse

Active Transport (NPC)

Large molecules require NLS/NES and transport receptors; energy-dependent and selective

Nuclear Localization Signal (NLS)

Short positively charged amino acid sequence directing proteins into nucleus (“VIP badge”)

Importin (Nuclear Import Receptor)

Binds NLS-containing proteins and transports them through NPC; releases cargo via Ran-GTP

Adaptor Protein

Links cargo to receptor when direct binding is not possible (“translator/connector”)

Ran-GTPase Gradient

Nucleus: high Ran-GTP; Cytoplasm: high Ran-GDP; drives directional transport

Ran-GEF

Located in nucleus; converts Ran-GDP → Ran-GTP

Ran-GAP

Located in cytoplasm; converts Ran-GTP → Ran-GDP

Nuclear Import Mechanism

Cargo + importin enter nucleus; Ran-GTP binds importin → cargo released; importin-Ran-GTP returns to cytoplasm

Nuclear Export Mechanism

Exportin + cargo + Ran-GTP exit nucleus; Ran-GTP hydrolysis releases cargo in cytoplasm

Ran Recycling

Ran-GDP returns to nucleus; Ran-GEF converts it back to Ran-GTP

Ran System Importance

Spatial separation ensures directionality; without it transport would be random

Endosymbiosis Theory

Mitochondria originated from engulfed aerobic bacteria forming mutualistic relationship

Mitochondrial Function (Symbiosis)

Bacteria produced ATP; host provided protection and nutrients

Mitochondria Evidence: Double Membrane

Inner membrane resembles bacterial membrane; outer membrane from host

Mitochondria Evidence: DNA

Contains circular DNA similar to bacteria

Mitochondria Evidence: Ribosomes

Contain 70S ribosomes and synthesize some proteins

Mitochondria Evidence: Division

Divide by binary fission

Mitochondria Evidence: Antibiotics

Sensitive to antibiotics targeting bacteria

Symbiosis Example

Kwang Jeon experiment showed amoebae dependence on bacteria

Symbiosis Example

Paramecium bursaria hosts symbiotic organisms for mutual benefit

Mitochondria Functions

ATP production, fatty acid β-oxidation, apoptosis, calcium storage, heat production

Outer Mitochondrial Membrane

Smooth, permeable via porins

Inner Mitochondrial Membrane

Highly selective; contains ETC and ATP synthesis machinery

Cristae

Folded inner membrane increasing surface area

Intermembrane Space

Stores protons during respiration

Matrix

Contains enzymes, mtDNA, ribosomes

Mitochondrial Protein Origin

Most proteins made in cytosol and imported; mitochondria make only some proteins

Mitochondrial DNA Significance

Maternally inherited; used in evolutionary studies; mutations cause disease

Mitochondrial Signal Sequence

N-terminal, positively charged, amphipathic α-helix; directs proteins to matrix

TOM Complex

Outer membrane translocator; entry for all mitochondrial proteins

TIM23

Inner membrane; imports proteins into matrix

TIM22

Inner membrane; inserts membrane proteins

SAM

Outer membrane; folds β-barrel proteins

OXA

Inner membrane; inserts mitochondria-made proteins

Mitochondrial Import Type

Post-translational; proteins imported fully synthesized and unfolded

Chaperones in Import

Hsp70 keeps proteins unfolded for transport

Energy for Import

Requires ATP and inner membrane proton gradient

Mitochondrial Import Steps

1. Protein synthesized 2. Hsp70 keeps unfolded 3. Enters TOM 4. Passes TIM23 5. Signal cleaved 6. Folds in matrix

Energy Requirements for Import

ATP (cytosol), ATP (matrix), proton gradient

Inner Membrane Protein Targeting

Stop-transfer sequence halts translocation and inserts protein into inner membrane

Intermembrane Space Protein Targeting

Partial import and cleavage releases protein into intermembrane space

Porin Insertion

Proteins go through TOM → SAM complex → inserted into outer membrane as β-barrel

Peroxisome Functions

Detoxification, fatty acid breakdown, H₂O₂ metabolism, lipid synthesis

Peroxisome vs Mitochondria Similarities

Both perform oxidative metabolism and import proteins from cytosol

Peroxisome vs Mitochondria Differences

Peroxisomes lack DNA and import folded proteins; mitochondria have DNA and import unfolded proteins

Peroxisomal Targeting Signal

SKL sequence at C-terminus directs proteins to peroxisome

Pex5 Receptor

Binds SKL signal and transports protein to peroxisome

Peroxisome Import Mechanism

Protein + Pex5 dock; temporary pore forms; protein enters folded; ATP required to release and recycle receptor

Zellweger Syndrome Cause

Mutation in PEX genes causing defective protein import

Zellweger Syndrome Effects

Empty peroxisomes, neurological defects, liver/kidney dysfunction, developmental issues

Zellweger Symptoms

Hypotonia, feeding issues, vision/hearing loss, seizures, distinctive facial features

Peroxisome Statement 1

True: all proteins encoded in nucleus

Peroxisome Statement 2

False: peroxisomes do not contain DNA

Peroxisome Statement 3

True: proteins imported post-translationally and folded

Topological Equivalence Example

Interior of nucleus is equivalent to cytosol (transport via pores, no membrane crossing)

Mitochondrial Import Question Answer

Correct: FFTT (transport driven by H⁺ gradient and N-terminal signal cleavage are true)

Ran-GTP Question Answer

Ran-GTP binding to import receptor causes release of cargo in nucleus

ER Structure

ER is a netlike labyrinth of branching tubules and flattened sacs extending throughout the cytosol; ER and nuclear membrane form a continuous sheet enclosing a single internal space (ER lumen)

ER Continuous Membrane Network

ER is a single interconnected membrane system continuous with the nuclear envelope (physically connected to nucleus)

Smooth ER (SER) Function

SER is the primary site of vesicle budding (transitional zones); involved in lipid metabolism and steroid synthesis (e.g., testosterone production in Leydig cells)

Smooth ER Structure

No ribosomes; tubular, smooth, irregular, and highly branched network

Rough ER (RER) Function

RER is site of protein synthesis, maturation, and transport

Rough ER Protein Types

Synthesizes integral membrane proteins and soluble secreted proteins

Rough ER Structure

Studded with ribosomes

Microsomes

Fragments of ER that reseal into closed vesicles; retain ER functionality and used experimentally

Blobel Experiment Question

How do proteins know where to go?

Blobel Experiment Step 1

Without ER: protein synthesized is longer than expected

Blobel Experiment Step 1 (With ER)

With ER: protein is shorter due to removal of signal sequence during entry

Protease Protection Experiment

Proteases digest proteins outside ER but not inside; proves proteins are translocated into ER lumen