Module 5 BIOL 2021

Endomembrane system

Network of membrane-enclosed organelles in eukaryotic cells (ER, Golgi, lysosomes, endosomes and more) that likely began with an expansion of the plasma membrane

Cytosol (function)

Contains many metabolic pathways, protein synthesis, and the cytoskeleton

Nucleus (function)

Contains the main genome; site of DNA and RNA synthesis

Endoplasmic reticulum (ER) function

Synthesis of most lipids; synthesis of proteins for distribution to many organelles and to the plasma membrane

Golgi apparatus (function)

Modification, sorting, and packaging of proteins and lipids for secretion or delivery to another organelle

Lysosomes (function)

Intracellular degradation

Endosomes (function)

Sorting of endocytosed material

Mitochondria (function)

ATP synthesis by oxidative phosphorylation

Chloroplasts (function)

ATP synthesis and carbon fixation by photosynthesis (in photosynthetic cells)

Peroxisomes (function)

Oxidative breakdown of toxic molecules

How the endomembrane system likely evolved

Plasma membrane protrusions formed internal membranes around archaeal DNA; an aerobic bacterium was engulfed (endosymbiont) and shed its encasing membrane; internal membranes pinched off to form the nucleus and elaborate the endomembrane system

Endosymbiont

Aerobic bacterium engulfed by an ancestral archaeon that became the mitochondrion; it kept its own genome

Three ways proteins enter organelles

1) Transport through nuclear pores 2) Transport across membranes (protein translocators) 3) Transport by vesicles

Where does translation occur?

In the cytosol on ribosomes (proteins for the ER are made by ribosomes that dock at the ER); proteins are then sorted to their destinations

Transport through nuclear pores

Mechanism 1: proteins move between the cytosol and the nucleus through nuclear pore complexes

Transport across membranes

Mechanism 2: proteins cross organelle membranes through protein translocators into the ER, mitochondria, chloroplasts, and peroxisomes

Transport by vesicles

Mechanism 3: proteins move between the ER, Golgi, endosomes, and the plasma membrane in budding and fusing vesicles

Gunter Blobel

Rockefeller University; 1999 Nobel Prize in Physiology or Medicine for discovering that proteins have intrinsic signals that govern their transport and localization in the cell

Sorting signal (signal sequence)

A stretch of amino acids written in the protein sequence that directs the protein to its final destination

ER targeting signal

N-terminal signal sequence for import into the ER (rich in hydrophobic amino acids)

ER retention signal

C-terminal KDEL (Lys-Asp-Glu-Leu); keeps a protein in the lumen of the ER

KDEL

C-terminal ER retention signal (Lys-Asp-Glu-Leu)

mTP (mitochondrial transit peptide)

N-terminal signal for import into mitochondria; contains positively charged amino acids such as Arg and Lys

NLS (Nuclear Localization Signal)

Signal for import into the nucleus; basic (positively charged Lys and Arg), e.g., Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val

NES (Nuclear Export Signal)

Signal for export from the nucleus; contains key hydrophobic amino acids (e.g., Leu, Met, Phe)

PTS (Peroxisome Targeting Signal)

C-terminal signal SKL (Ser-Lys-Leu) for import into peroxisomes

SKL

C-terminal peroxisome targeting signal (Ser-Lys-Leu)

Which signals are N-terminal vs C-terminal?

N-terminal: ER targeting signal, mTP/cTP transit peptides. C-terminal: ER retention signal (KDEL) and PTS (SKL)

Nuclear envelope

Double membrane (inner and outer nuclear membranes) around the nucleus; continuous with the ER and perforated by nuclear pores

Nuclear pore complex (NPC)

Large multiprotein channel in the nuclear envelope that selectively transports proteins in and out of the nucleus

Small proteins and the NPC

Small proteins can diffuse through the nuclear pore into the nucleus

Parts of the NPC (from the figure)

Cytosolic fibrils, nuclear basket, and nuclear pore complex proteins spanning the nuclear envelope

Nuclear lamina

Protein meshwork lining the inside of the inner nuclear membrane

2025 yeast NPC structures

Cell (2025) paper reported the comprehensive structure of the yeast nuclear pore complex, both isolated and in situ

Importin

Nuclear import receptor that recognizes proteins carrying an NLS

Importin alpha

Subunit of the import receptor that recognizes the NLS on the cargo protein

Importin beta

Subunit that interacts with the nuclear protein/importin alpha and with the NPC

Ran

Ras-related nuclear protein; a GTP-binding protein (GTPase) that drives nuclear transport

Ran-GEF

Guanine nucleotide exchange factor for Ran; promotes exchange of GDP for GTP, producing Ran-GTP (in the nucleus)

Ran-GAP

GTPase-activating protein for Ran; stimulates hydrolysis of Ran-GTP to Ran-GDP (in the cytoplasm)

Energy source for nuclear transport

GTP hydrolysis by Ran

Ran cycle step 1

A protein with an NLS binds importin in the cytoplasm and enters the nucleus through the nuclear pore

Ran cycle step 2

In the nucleus, Ran-GTP binds importin, causing release of the nuclear protein

Ran cycle step 3

Importin-Ran-GTP is exported back to the cytoplasm

Ran cycle step 4

Ran-GAP hydrolyzes Ran-GTP to Ran-GDP, releasing importin for another cycle

Why does importin release its cargo in the nucleus?

Ran-GTP (high in the nucleus) binds importin and causes release of the nuclear protein

Transit peptide

N-terminal signal sequence that directs proteins to mitochondria (mTP) or chloroplasts (cTP)

What happens to mTP and cTP after import?

They are cleaved, so mature mitochondrial and chloroplast proteins are smaller than their precursors

Energy requirement for mitochondrial import

Import needs energy, including the H+ gradient across the inner mitochondrial membrane

Precursor protein

Newly made protein that still carries its signal sequence before import

Import receptor protein (mitochondria)

Outer membrane protein that binds the signal sequence of the precursor protein

TOM

Protein translocator in the outer mitochondrial membrane

TIM

Protein translocator in the inner mitochondrial membrane

Steps of mitochondrial protein import

Signal sequence binds import receptor, protein passes through TOM and TIM into the matrix, and the signal peptide is cleaved to give the mature protein

MitoTracker

Fluorescent dye that binds thiol-reactive chloromethyl groups in the mitochondrial membrane; used to mark mitochondria

Co-localization

Overlap of two fluorescent signals in a merged image (e.g., GFP fusion plus MitoTracker looks yellow), suggesting the protein is in that organelle

How to tell a GFP fusion protein is in mitochondria vs chloroplasts

Mitochondria: GFP merges with MitoTracker. Chloroplasts: GFP merges with chlorophyll autofluorescence

Peroxisomal import signal

Peroxisome proteins carry a C-terminal PTS (SKL)

ER signal sequence location

N-terminus; the ER retention signal (KDEL) is at the C-terminus

Effect of adding or removing an ER signal sequence

It can redirect the localization of any protein

SRP (Signal Recognition Particle)

Recognizes the ER signal sequence on the growing polypeptide and brings the ribosome to the SRP receptor on the ER

SRP receptor

ER membrane protein that docks the SRP-ribosome complex

Protein translocator (ER)

Channel in the ER membrane that receives the growing polypeptide and passes it across the membrane

After the ribosome docks at the ER

The SRP is displaced and released for reuse, and the growing polypeptide is transferred into the translocator

Signal peptidase

ER enzyme that cleaves the N-terminal signal peptide inside the ER lumen

Fate of a soluble ER protein

It crosses the ER membrane into the lumen, the N-terminal signal peptide is cleaved, and the mature protein stays in the lumen

Stop-transfer sequence

Hydrophobic sequence (transmembrane domain) that stops translocation and anchors the protein in the ER membrane

Single-pass transmembrane protein

Made when an N-terminal ER signal sequence is followed by one hydrophobic stop-transfer sequence

Start-transfer sequence

Internal hydrophobic sequence that starts translocation; together with stop-transfer sequences it produces multipass membrane proteins

Multipass membrane protein

Produced by alternating internal start-transfer and stop-transfer sequences

Transport vesicle

Small membrane-bound sac that carries membrane components and soluble proteins by budding from one membrane and fusing with the next

Vesicular transport

Cargo moves between compartments by vesicles budding from one membrane and fusing with another

Clathrin

Coat protein that forms basket-like cages to help shape the membrane into vesicles; does NOT select cargo

Adaptin

Adaptor protein that binds clathrin and selects cargo molecules

Dynamin

GTPase that pinches off the neck of the budding vesicle

Clathrin-coated vesicle formation steps

Coat assembly and cargo selection, bud formation, vesicle formation (dynamin pinches off), then uncoating to a naked transport vesicle

Uncoating

Removal of the clathrin coat after budding so the vesicle can dock and fuse

Rab

Small GTP-binding protein on vesicles that helps with tethering to ensure the vesicle goes to the right membrane

Tethering protein

Protein on the target membrane that captures a vesicle bound by Rab

v-SNARE

Vesicle SNARE on the transport vesicle

t-SNARE

Complementary target-membrane SNARE on the target membrane

Steps of vesicle docking and fusion

Tethering (Rab + tethering protein), docking (v-SNARE pairs with t-SNARE), fusion (membranes coalesce and lipid bilayers fuse), cargo delivered

What if clathrin, adaptin, or dynamin is missing?

Vesicles cannot be properly formed or pinched off, so cargo is not packaged and delivered (expected result)

What if Rab or SNAREs are missing?

Vesicles cannot dock and fuse with the correct target membrane, so cargo is not delivered (expected result)

Nobel Prize 2013 (Physiology or Medicine)

Rothman, Schekman, and Sudhof for discoveries of the machinery regulating vesicle traffic

Exocytosis

Vesicles fuse with the plasma membrane to release contents outside the cell; the secretory pathway to the cell surface

Glycosylation in the ER

Covalent attachment of a short, branched oligosaccharide to an asparagine (Asn) residue of a protein

ER quality control

The ER checks protein folding: chaperones help folding, and misfolded proteins are removed

Chaperones

Proteins in the ER lumen that help proteins fold correctly

ERAD

ER-associated degradation; pathway that degrades misfolded proteins from the ER

UPR (unfolded protein response)

Signaling pathway triggered by accumulation of misfolded proteins in the ER lumen

Cisterna (plural cisternae)

Membrane-enclosed sac; the flattened compartments that make up the Golgi

Order of Golgi compartments

ER -> cis Golgi -> cis cisterna -> medial cisterna -> trans cisterna -> trans Golgi (network)

Golgi apparatus (modification)

Proteins are further modified in the Golgi apparatus as they move through the cisternae

Trans Golgi network

Where the constitutive and regulated secretion pathways diverge

Constitutive secretion

Unregulated exocytosis: continuous delivery of newly synthesized plasma membrane proteins and lipids and soluble proteins to the cell surface

Regulated secretion

Secretory vesicles store concentrated proteins and release them by exocytosis only after an extracellular signal (hormone or neurotransmitter) triggers signal transduction

Phagocytosis

Cell takes in large particles (e.g., bacteria, old cells) and encloses them in phagosomes; done by immune cells

Phagosome

Vesicle that encloses a large particle taken in by phagocytosis

Neutrophil

Immune cell that engulfs bacteria; neutropenia is a disease with too few neutrophils