Genetic Biochemistry - Lecture 1

Intracellular compartments

· in aqueous environ., lipid has polar head exposed, tries to bury hydrophobic tail

· lipid bilayer [1 Lewin 2.4]

· in diff. membranes, lipid composition varies considerably

· both ratio of protein to lipid

· types of lipid

· tf; diff. membranes have diff. biophysical properties

· imp. property of membrane, lipids can move laterally (not between layers) - fluidity

· more readily tails of adj. lipids pack, more crystalline structure (less fluid) ?

· depends on length and type of lipid tails which more f lui SD -

· unsaturated chains more difficult to pack, more fluid

· PM of animal cells relatively large anits cholesterol - increases

mechanical stability

· plant cells lack cholesterol, but have other sterols

· PM only membrane to contain signif. amt glycolipids

· consider membranes as lipid bilayer to provide residence for large protein

molecules [2 Lodish 14-1]

· proteins can move laterally, but difflise more slowly than lipid

· proteins can be internalized due to stimulus, others pass thru

membrane as secreted, tf; proteins associated with bilayer dynamic

· transmembrane protein (e.g., receptors)

· membrane has two "faces"

· cytosolic face

· noncytosolic (called diff. names depending where it is)

· PM, extracellular environment

· organelle, lumen

· uneven distribution of three components [2 Lodish 14-29]

· (1) diff. lipids in diff. monolayers (1,oth polar head group and tails)

· lipids on cytoplasmic face more highly charged, tend to be unsat

(more 2

· PM, exterior of cell rich in oligosaccharides (cell adhesion, cell-cell recog.) · Compartmentalization [3 Alberts 12-1]

· little more than half total volume of cell

· site of protein synthesis

· most of intermediary metabolism (small molecule degradation and synthesis

to provide building blocks of macromolecules)

· ER

· about half total area of membrane

· membrane bound ribosomes, synthesis of integral membrane proteins and

soluble proteins (most destined for secretion or other organelles)

· this is important difference biw how proteins directed to ER and other

cytoplasmic organelles

· proteins translocated to ER during synthesis, to other organelles only

after synthesis complete

· produces lipid for rest of cell

· flinctions as store for Ca²+

more RER · Topological Relationships

A/V=10.7 A/V=3.4

· most proteins do not have sorting signal, remain in cytosol

· others have specific signals for nucleus, ER mito, plastids, peroxisomes

· can also sort from ER to other destinations in cell

· 3 fundamental ways for movement from one compartment to another

· (1) gated transport - protein traffic biw cytosol and nucleus (topo equiv spaces) in

continuity through nuclear pore complexes [3 Alberts 12-4]

· (2) transmembrane transport - membrane-bound protein translocators directly

transport specific proteins across a membrane from cytosol to space topo distinct

· transported protein must unfold to go through membrane

· initial transport of proteins from cytosol into ER lumen or mito

· nuclear localization signals (NLS)

(present only in nuclear proteins)

· defined by recombinant DNA technology - domain swap (explain)

· normally accumulates in nucleus shortly after cyto syn

· single aa mutation prevented nuclear import

· used short lengths of DNA encoding region around mutation to define

localization signal fused to cytosolic protein

· Active transport

· initial interaction reqs one or more cytosolic proteins that bind to NLS, help

direct nuclear protein to pore complex (appears to bind to fibrils that project

from rim of complex)

· nuclear protein moves to center of pore complex, actively transported by

process that requires ATP

· using various sized gold beads, opening can dilate ii~

·

· Export of new ribosomal subunits and mRNA also depends on selective transport

· 20-nm dia gold spheres coated wi small RNA molecules (5S or tRNA),

injected into nucleus (frog oocyte), rapidly transported through nuclear pores

into cyto

· if injected into cyto, remain there; tf; seems pore contains receptors that

recognize RNA molecules (or proteins bound to them) destined for cyto

· ER

· transmembrane proteins, only partly translocated across ER membrane, become

embedded in it

· some will remain in ER, many destined to reside in PM or membrane of

another organelle

· water soluble proteins, fully translocated, released into lumen of ER

· destined for lumen of organelle or for secretion

· all these proteins, regardless of fate, directed to ER membrane by same kind of signal peptide

and translocated by same mechanism

· import begins before polypeptide chain completely syn - co-translationally

· different than import into mito, chloroplasts, nuclei, peroxisomes - posttranslational

and requires different signal

· never released into cytosol; tf, never folds before reaching translocator in membrane

· in contrast, posttranslational import into mito, chioro, cytosolic chaperones required

to keep unfolded

· ribosome synthesizing protein directly attached to ER membrane - create regions

termed RER

· SRP ribosome complex binds to SRP receptor, integral membrane protein (2

subunits) exposed on cyto surface of RER

· Sequence of events

· signal seq cleaved off in lumen by signal peptidase, quickly degraded

· peptide chain continues elongate, extruded through ER mem

· Bip binds to exposed hydrophobic segments

· prevent denaturation or aggregation

· ATP hydrolysis drives Bip release

·

OK

propcrly, not

· ~~aiibe of to

· quality checkpoint for proper folding and assembly (don't want misfolded proteins to

reach cell surface, may be recognize as foreign, might stimulate immunologic

response

· tf, ER one of maj or sites where proteins degraded

· Golgi apparatus [10 Alberts 13-4]

· two distinct faces (cis - entry face, trans - exit face)

· both closely connected to special compartments - network of interconnected

tubularstructures

· cis compartment thought continuous wi CGN

· next compartment, medial - central cisternae of stack

· trans compartment, final site of glycosylation - lumen thought continuous w/TGN · Oligosaccharide processing in Golgi

· two broad classes of N-linked oligo [11 Lodish 16-27]

· high-mannose oAligosaccharides

· no new sugars added in Golgi

· contain 2 GlcNAc, many mannose residues

· compAlex oAligosaccharides

· more than original 2 GlcNAc, variable number galactose, sialic acid

(only sugar in glycoproteins winet negative charge), and sometimes

fucose

· formed by combination of trimming of original oligo and addition of

other sugars

· which processing determined mainly by configuration of protein

· if oligo on protein accessible to enzymes in Golgi, then likely converted to

complex form

· protein may fold rapidly, so site not accessible

· if inaccessible, likely remain high-mannose form

· each cell type has its own set processing enzymes, thus same protein may be

modified differently in different cells

· processing follows highly ordered pathway [11 AAlberts 13-11]

· each cisterna contain own set of processing enzymes

· proteins modified in successive stages as move from one cisterna to next

· only accepted as substrate if properly processed by preceding enzyme

· forward movement mediate by transport vesicles

· Vesicular Transport

· fusion of vesicles w/PM called exocytosis

· hormones, neurotransmitters, digestive enzymes · Endocytosis

· large vesicles (phagosomes), general> 250 nm

· imp in animal cells for purposes other than nutrition

· normally specialized phagocytic cells

· mammals, two classes wbc "professional" phagocytes

· macrophage - widely distributed in tissues and blood

· neutrophils

· defend against infection, digest invading microorganisms

· macros also scavenge senescent, damaged cells, debris

· quantitatively more imp

· phagocytose> 1011 senescent rbc each day

· pinocytosis - ingestion of fluid and solutes

· small vesicles (<=150 nm)

· extracellular fluid trapped, substances dissolved in fluid internalized - called

fluid-phase endocytosis

· most of their PM

· by cell

· e g , ingest 25% own volume of fluid/hour

· 3% Iniri, or 100% ~ 30

and vesicles, extend from periphery to perinuclear region (12 Alberts 13 -3)

· often close to Golgi, but distinct

· two set of endosomes readily distinguished

· earAly endosomes -just beneath PM

· tracers appear within a minute or so

· Alate endosomes - close to Golgi and near nucleus

· tracer appears after 5-15 min

· interior of endo kept acidic (pH 6) by driven H+

· than

· early endo acts as main sorting station (like TGN in exo pathway)

· many ligands released in acidic environment

· other ligands remain bound, share fate wireceptor

· (1) most receptors return to PM domain they came from

· LDL receptor

· receptor recycled to PM

· LDL goes to lysosome

· transferrin receptor pathway similar, except transferrin

stays bound to receptor, releases iron in endosome,

returns to PM, released in neutral pH of extracellular

fluid

· (2) some go to lysosome for degradation

· EGF receptor clusters in pits only after binding ligand

· most do not recycle, degraded in lysosome

Directed Searches:

Golgi vesicles: Think of Golgi bodies as protein post offices preparing proteins for transport.
Nuclear Membrane

Lysosome: The cellular composter, breaks down waste materials to their components, pH=5.

Secretory vesicle: Organelles responsible for hormone et. al. transport via exocytosis. Ref A. Ref B.

Mitochondrion: This double membrane organelles are the batteries of the cell. Convert glucose and oxygen to ATP. Krebs cycle and glycolysis pathways are active within mitochondria.

Endoplasmic Reticulum: A three dimensional maze where:

a) large molecules are transported
b) proteins are stored
c) ribosomes are attached
d) fatty acids and lipids are synthesized.

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