Evolution of Internal Membranes of Eukaryotes
- precursors of eukaryotes believed to be organisms (like bacteria) with no internal membranes
- plasma membrane carried out all membrane-related functions
endomembrane system thought to have evolved as invagination of plasma membrane - mitochondria and chloroplasts thought to have evolved as endosymbionts
Three Ways to Sort Proteins in Eukaryotic Cells
Protein sorting = transfer of proteins into compartments where they are needed
* synthesis of virtually all proteins starts in cytosol, on free ribosomes
* All protein transport requires energy
1. Transport through nuclear pores
2. Transport across membranes
3. Transport by vesicles
Signal Sequence
- stretch of amino acids, at N-terminus, 15-60 amino acids long, that directs proteins to particular organelles
- Signal sequences for nucleus, mito/chloro, peroxisomes or ER
- Usually removed after sorting
- Delete or transfer sequence to another protein — protein goes to wrong ‘address’
Nuclear Pore Complex: highly selective gate
- very high traffic through nuclear pores (500 molecules through each of the 3000-4000 per second) but flow is SELECTIVE
- Proteins to be imported have nuclear localization sequence
Nuclear Pore Complexes — Gateways of the Nucleus
- small molecules (even small proteins) freely pass through nuclear pores
- Passage of larger proteins is active (energy-requiring) process
- Nuclear localization signal - amino acid sequence that ‘tags’ a protein for import into the nucleus by nuclear transport
- nuclear export signal tags a protein for export
- Proteins pass through nuclear pore complexes without unfolding
What moves out of nucleus?
- mature, properly processes mRNA
- Ribosomal RNA (manufactured in nucleolus)
What moves into nucleus?
- histones, proteins required for transcription and DNA replication
- dNTPs, rNTPS
Transport across Membranes: Mitochondria
- mitochondria and chloroplasts have a double membrane — chloroplasts have a third membrane (thylakoids)
- although they have their own genomes and ribosomes, most of their proteins are encoded by nuclear genome so it must be imported
Transport across Membranes: Mitochondria pt 2
- Proteins destined for mitochondria/chloroplasts made by free ribosomes in the cytosol
- signal sequence at N terminus
- proteins must be moved across both outer and inner
- membranes at special sites where layers are in contact
- proteins must unfold to be imported, then refold and signal sequence removed
- Subsequent transport within organelle requires another signal sequence (exposed after first one removed)
Transport across Membranes: Endoplasmic Reticulum
- ER is most extensive of Endomembrane system
- Serves as an entry point for not only proteins for the ER itself, but rest of Endomembrane system (golgi, lysosomes, endosomes), cell surface, secretory proteins
- Once in ER (in membrane or lumen), proteins will never re-enter cytosol
- Synthesis of all proteins starts on free ribosomes
Two types of proteins transferred to ER:
- water soluble proteins translocated completely across into ER lumen — destined for lumen of an organelle or secretion (out of cell)
- Prospective transmembrane proteins translocated only partially across — destined for plasma membrane, ER membrane or membrane of another organelle
____ and ____ direct ribosome to ER
ER signal sequence and an SRP
Vesicular Transport
Temporary vesicles:
* allow material to leave and enter cells
* Move material between Endomembrane compartments
* Carry soluble proteins (in their lumens) to the plasma membrane for secretion
* Move membrane proteins (in their membranes) to be expressed on the cell surface
Vesicular Transport pt 2
with respect to proteins:
transport vesicles carry soluble proteins (in their lumens) and membrane proteins (in their membranes) between compartments
in general vesicle traffic is …
- outward from ER: Golgi — other organelles? Plasma membrane?
- Inward: plasma membrane — endosomes — lysosomes
Vesicle budding is driven by
formation of protein coat
Clathrin-Coated Vesicles
- mediate transport from outward face to golgi and inward from plasma membrane
- Clathrin forms ‘basket’ that gives vesicle shape
- ‘Adaptins’ capture specific cargo molecules by trapping the receptors that bind to them
Outward Flow of Traffic within Endomembrane System
Rough ER: synthesis of proteins for - export (secretion) - insertion into membranes - lysosomes Golgi apparatus: collection, packaging & distribution
Steps along the Secretory Pathway: ER Processing
most proteins are covalently modified in ER
1. formation of disulfide bonds
- stabilize protein shape
2. addition of sugar groups – glycosylation
various functions depending on protein …
- protect protein from degradation
- keep protein in ER until properly folded
- help direct to protein to proper organelle (act as transport signal for packaging into appropriate vesicles)
- if displayed on cell surface, cell-cell recognition
Glycosylation in the ER
as growing peptide enters ER, ‘prefab’ carbohydrate group attaches to amino (NH2) groups of asparagine (Asn) side chains
“N-linked glycosylation”
Only properly folded proteins are allowed to leave ER
“Unfolded Protein Response” (UPR)
- if protein production exceeds capacity to keep up with folding — misfolded proteins accumulate
- Signals lead to increased expression of chaperone proteins and other proteins that assist in folding, expansion of the ER …
- If cell still can’t keep up, UPR will trigger cell death — apoptosis
Steps along the Secretory Pathway: Modification and Sorting in the Golgi Apparatus
- series of flattened sacs - cisternae
- Organized into functionally distinct compartments with cis (entry) face closest to ER, trans (exit) face at other end
- cis — newly formed
- trans — breaking away
Functions of Golgi Apparatus: PTMs
- modification of new proteins arriving from ER:
- Peptide chains shortened by proteases
- Amino acids modified
- CHO groups that were added in ER modified or removed glycosylation
- different CHO groups added to different AAs (ser, thr)
“O-linked glycosylation “
- different CHO groups added to different AAs (ser, thr)
- Most complex polysaccharides are synthesized in the golgi
- Glycos amino glycans in extracellular matrix (animals)
- Pectins, hemicellulose (plant cell walls)
Vesicular Transport and Endocytic (Inward) Pathways
- taking substances into cell by surrounding them with membrane
- they become a membrane-bound vesicle
