Module 12 - the System of Internal Membranes (Secretion)

included on exam 3

endocytosis

import mediated by the formation of endocytic vesicles

the taking in of other things into the cell (endo, into)

exocytosis

export (secretion) out of a cell

exchange of materials between cell and environment

endocytosis and exocytosis

involves the plasma membrane, small vesicles, endoplasmic reticulum, Golgi apparatus, and lysosomes

peroxisomes

one of the digestive organelles of a eukaryotic cell

contain enzymes that break down lipids and destroy toxic molecules through oxidation reactions

(perOXIsomes, OXIdative breakdown)

eukaryotic nucleus

the center of a eukaryotic cell

site of DNA storage and replication

consists of nuclear envelope (and ER?), perinuclear space, and nuclear pores

nuclear envelope

a double membrane surrounding the nucleus

(made of outer and inner nuclear membranes)

outer membrane is continuous with the ER

perinuclear space

the cavity in between the two membranes of the nuclear envelope

nuclear pores

holes in the nuclear envelope that allow both passive and active transport

contain protein complexes that facilitate transport through the nuclear envelope (e.g. cytosolic fibrils, nuclear basket)

nuclear lamina

a network of lamin protein fibers that lines the inner wall of the nuclear envelope

provides structural support for nucleus

provides attachment sites for chromatin

when destroyed, the entire nucleus collapses

a component of cytoskeleton (categorized as intermediate filaments)

(green in the picture)

passive diffusion through nuclear pores

for small molecules and small proteins (<50 kDalton)

no energy required

left side of the picture

active transport through nuclear pores

required for larger proteins and all RNAs

energy-dependent

right side of the picture

nuclear localization signal (NLS)

an amino acid sequence that is included in proteins that are transported through the nuclear envelope

allows a protein to enter the nuclei of cells

(e.g. included in DNA polymerases, RNA polymerases, transcription factors, histones, other chromatin assembly proteins, lamins, SV40 T-antigen)

the SV40 T-antigen

contains an NLS: 7 amino acids in the middle of the protein

oncogenic; capable of altering nuclear transcription factor activity of cells → transforming those cells

NLS allows it to enter the nuclei of infected cells, cause malignancy

if a protein (e.g. pyruvate kinase) lacking an NLS is given the SV40 T-antigen NLS as an expression vector, pyruvate kinase still fails to translocate to the nucleus (? not sure on this one)

process of active transport through a nuclear pore

transport complex forms, containing cargo protein and nuclear transport receptor protein

“cargo” protein: nuclear protein with an NLS

nuclear transport receptor protein: recognizes and binds to NLSs in cargo protein

translocation cycle:

entire transport complex is translocated through pore by interactions with pore proteins (e.g. cytosolic fibrils)

Ran-GTP binds to the nuclear transport receptor protein → causes dissociation and release of cargo protein inside nucleus

Ran-GTP with nuclear transport receptor protein goes to cytoplasm

ran GTPase activating protein (Ran-GAP) removes a P_i from the GTP on Ran

nuclear transport receptor protein dissociates from Ran-GDP, returns to the cycle

the Ran-GTP gradient

important for active transport through a nuclear pore

Ran-GTP is more abundant inside nucleus → will tend to go to cytoplasm

in cytoplasm, GTP is hydrolyzed to GDP by Ran GAP

→ Ran-GTP gradient is maintained across nuclear envelope → its diffusion drives the nuclear import cycle

energy-dependent: requires GTP hydrolysis

Ran-GDP produced in cytoplasm goes back to nucleus with its own concentration gradient

GDp on ran is exchanged for GTP (catalyzed by Ran-GEF)

regulation of import of NF-KB

NF-KB is dormant in cytoplasm with IKB bound to it

IKB masks the NF-KB’s NLS → prevents importin from binding to it

if IKB is phosphorylated and destroyed, NF-KB is released → importin can bind, translocate it to the nucleus so it can activate transcription

NF-KB is a transcription factor with a key role in regulating immune response to infections

regulation of import of Pho4

Pho4: a transcription factor, exists in cytoplasm in an inactivated phosphorylated state

phosphate groups block NLS

if dephosphorylated, importin can bind to it and take it to the nucleus

involved in regulation of the cell division cycle

smooth endoplasmic reticulum (ER)

the site of lipid synthesis and processing

builds membranes and membrane systems for the cell

contains no ribosomes

rough ER

site of protein synthesis and processing

site of protein folding, addition of cofactors, lipids, and carbohydrate groups

association of ribosomes → rough appearance

translation

polypeptide synthesis by ribosomes in the cytoplasm

occurs in a free ribosome if a synthesized protein is meant to stay in the cytoplasm, go into the nucleus, chloroplast, mitochondria, or peroxisome

occurs in rough ER ribosomes if the synthesized protein is a membrane protein, lysosomal, or secreted

^translocated into the lumen of the ER right after synthesis

synthesis of secreted proteins

all secreted proteins contain an ER signal sequence in their amino terminal

signal is necessary for translocation into ER; gets removed after translocation

ER signal sequence is recognized by SRP (signal recognition particle)

SRP docks on SRP receptor on ER membrane

growing polypeptide moves through translocation channel

signal peptidase removes translocation signal from the new polypeptide

secretion pathway

path taken by proteins synthesized on the rough ER

ER for protein processing → ERGIC for retrieval of ER-resident components → Golgi apparatus for protein sorting

from Golgi, can go to:

out of cell through secretory vesicles (secreted proteins), of

to plasma membrane (membrane proteins), or

to lysosomes (lysosomal proteins)

the pulse-chase experiment

a way to track the secretion pathway

steps:

1) incubate cultured pancreatic cells with ³⁵S-labeled amino acids for 3 minutes (pulse: all proteins synthesized during that time will be radioactive)

2) remove radioactive medium (no more radioactive protein synthesis)

3) follow movement of radioactive proteins by periodic autoradiography (chase: migration of radiolabeled proteins is tracced following radioactive signal)

ERGIC

ER-Golgi intermediate complex

a system of membrane stacks located between the ER and the Golgi

function: a checkpoint to ensure that ER-resident proteins do not reach the Golgi

reason: traffic from ER to Golgi is non-selective

ER-resident proteins contain a KDEL-sequence retention signal

if they reach the ERGIC, their are recognized, retrieved, sent back to ER

(blue in the picture)

Golgi apparatus

the protein sorting center of the cell (post office)

functions: protein modification, sorting, and packaging for secretion or delivery to other organelles8

protein-containing vesicles coming from ER enter at the cis face of the Golgi

proteins are moved through medial and trans Golgi stacks and released in vesicles from the trans face

3 types of transport from the Golgi

constitutive

regulated

lysosomal

constitutive transport from the Golgi

operates in all cells

unregulated secretion

also includes delivery of membrane proteins and lipids

(blue in the picture)

regulated transport from the Golgi

operates only in specialized cells

cells release their secreted products only when stimulated by a signal

signal is usually external, sometimes internal

products to be secreted are sorted into special, large vesicles that wait in the cell until the signal is received

exocytosis is calcium-dependent

e.g. endocrine cells secreting hormones, neurons secreting neurotransmitters

(red in the picture)

calcium-dependence of regulated secretion

fusion of secretory vesicles to cell membrane for release of products is induced by Ca²⁺

in simple endocrine cells: external signals cause opening of Ca²⁺ stores in ER

in neurons: extracellular Ca²⁺ inflow is observed in axon terminals (uncommon in simple endocrine cells)

synthesis and regulated secretion of insulin

preproinsulin starts with signal sequence, B segment, connecting C segment, and A segment

ER signal sequence is removed in the ER, disulfide bond forms

proinsulin: B and A disulfide-linked with C still attached

C segment is removed in secretory vesicle → insulin has just B and A

regulated by blood glucose: when high, insulin is released into extracellular space

lysosomal transport from the Golgi

transport of digestive proteins into the lysosome

all vesicles that target the lysosome are clathrin coated (either from Golgi or from the plasma membrane)

clathrin-coated vesicles

originate from trans-golgi (if carrying lysosomal enzymes) and plasma membrane (from endocytosis)

headed toward lysosomes

when lysosomal protein contacts inner surface of trans-Golgi, clathrin coat is assembled on other side

formation of coat mechanically induces vesicle formation

lysosomal proteins are recognized by receptors on the Golgi inner surface, contact of lysosomal cargo protein with receptor induces clathrin coat assembly)

dynamins assemble around the budding vesicle’s neck and pinch it off

vesicle is released, coat is shed (because clathrin is only needed for mechanically bending the membrane to form the vesicle)

lysosome

one of the digestive organelles of the eukaryotic cell

receives molecules/particles taken in by endocytosis, or cells taken in by phagocytosis, or obsolete/damaged cell components by autophagy for enzymatic degradation

contains acid hydrolases (digestive enzymes)

contains hydrolytic enzymes (not oxidative enzymes like peroxisomes)

needs a highly acidic environment inside to function (pH lowered by a H⁺ gradient) (pH~5 instead of pH~7.2 like in the cytoplasm)

components: early endosome, late endosome, maturation into functional lysosome

only forms as a result of its function

formation of the lysosome

early endosome forms from fusion of endocytic/phagocytic/autophagocytic vesicle

late endosome forms when hydrolase-containing vesicles from Golgi fuse with early endosome

late endosome matures into functional lysosome as pH decreases and hydrolytic enzymes become active

receptor-mediated endocytosis

taking in of specific molecules or particles recognized by cell surface receptors

e.g. endocytosis of cholesterol

endocytosis of cholesterol

an example of receptor-mediated endocytosis

cholesterol is blood-insoluble

transported from liver to bloodstream and around body bound to proteins → LDLs (low density lipoproteins)

LDL is recognized by surface LDL receptors, taken in by cell, delivered to lysosome

LDL is dissociated by digestive enzymes in lysosome, cholesterol is released for membrane synthesis

LDL receptors return to surface and get recycled

phagocytosis

the ingestion of other cells

done by specialized cells

e.g. a neutrophil ingesting a dividing bacterium

e.g. a macrophage ingesting damaged red blood cells

phagocytic cells have pseudopods: extensions of the membrane that extend over the ingested cell

autophagy

self-digestion

the process by which cells eliminate damaged or obsolete cell components

also occurs when cells are starved; cells “eat” their own components for food

4 steps: nucleation, closure, fusion, digestion

its existence was demonstrated in yeast cells by Yoshinori Ohsumi: mutant cells lacking degradation enzymes were starved, autophagosomes accumulated in the vacuole (see picture)

4 steps of autophagy, expanded

1) nucleation (recruitment of membrane and necessary molecules), extension of delimiting membrane

2) closure of double membrane autophagosome with its components

4) fusion of outer membrane with a lysosome

4) digestion of inner membrane and its contents