MB 207 – Molecular Cell Biology Intracellular compartments and protein sorting

Compartmentalization of cells Bacterium consists of a single intracellular compartment surrounded by plasma membrane. Eukaryotic cell is subdidvided into functionally distinct, membrane-enclosed compartments. Each compartment or organelle contains its own characteristic of enzymes and other specialized molecules, and complex distribution systems transport specific products from one compartment to another. → protein (enzymes, transporters, surface markers) 10 000 – 20 000 proteins are synthesized in the cytosol and delivered specifically to the cell compartment that requires it.

Major intracellular compartments of an animal cell → Vital biochemical processes take place in or on membrane surfaces. → Compartments increasing surface area as well as providing specialized aqueous spaces for reaction.

In most cells, Golgi apparatus is located close to the nucleus whereas the network of ER tubules extends from the nucleus throughout the entire cytosol. The localization of both ER and Golgi apparatus depends on an intact microtubule array.

The evolution of internal membranes: Development of plastid

Hypothetical scheme for the evolution of the cell nucleus and ER

Topological relationships between compartments of the secretory and endocytic pathways in eucaryotic cell Cycles of membrane budding and fusion permit the lumen of any of these organelles to communicate with any other and with cell exterior by means of transport vesicles. Arrows indicate the extensive network of outbound and inbound traffic routes.

A simplified ‘roadmap’ of protein traffic Gated transport: Gated system (nuclear pore complexes) to actively transport specific molecules. Transmembrane transport: Membrane-bound protein translocaters directly transport proteins across a membrane. Vesicular transport: Membrane-enclosed vesicles transport proteins from one compartment to another.

Three main mechanisms of protein transport B B C C

Vesicular transport: Vesicular budding and fussion

Summarizes the routes by which protein are carried forward or diverted to other organelles

General principles about protein trafficking Signal sequences and signal patches direct proteins to the correct cellular location - Signal sequence: signal resides in a single discrete stretch of amino acid sequence, often cleaved, not part of final protein product - Signal patch: 3-D arrangements of amino acids on the protein’s surface that forms when the protein folds up Proteins synthesized on free cytoplasmic ribosomes are imported post-translationally (e.g. nuclei, mitochondria, peroxisomes) and proteins synthesized on ER-bound ribosomes are imported co-translationally (e.g. ER) Proteins can fold before membrane translocation if translocation machinery can cope with large, folded protein structures (e.g. nuclei & peroxisomes) OR they will fold only after membrane translocation (e.g. mitochondria & ER).

Signal sequence and signal patch

The transport of molecules between nucleus and cytosol → Bidiectional traffic occurs continuously between cytosol and nucleus.

Nuclear pore complexes perforate the nuclear envelope composed of more than 50 nucleoporins, arranged in octagonal symmetry. more active the nucleus in transcription, greater the number of pore complexes. On average, each pore need to import 100 histone molecules/min and export 6 large and small ribosomal subunits/min.

Possible paths for free diffusion through nuclear pore complex Results from injection (molecules of different sizes) → <5000 daltons molecule: fast diffusion 17kD protein: 2 mins >60kD: cannot enter → channel is 9nm in diameter and 15nm long Nuclear envelope enables nuclear compartment and cytosol to maintain different complements of proteins. → eg. protein synthesis is confined to the cytosol.

Nuclear localization signals direct nuclear proteins to the nucleus Immunofluorescence micrographs showing T-antigen localization.

Nuclear import receptors bind nuclear localization signals and nucleoporins Many nuclear import receptors bind both to nucleoporins and to a nuclear localization signal on the cargo proteins they transport. Cargo proteins 1, 2 and 3 contain different nuclear localization signals, which causes each to bind to a different nuclear import receptor. Cargo protein 4 requires as adaptor protein to bind its nuclear import receptor. The adaptors are structurally related to nuclear import receptors and recognize nuclear localization signals on cargo proteins. They also contain a nuclear localization signal that binds them to an import receptor. Nuclear export receptors relies on nuclear export signals to transport macromolecules to cytosol.

The Ran GTPase drives directional transport Ran is required for both nuclear import and export systems. Ran is a molecular switch that can exist in two conformational states, depending on whether GDP or GTP is bound.

Ran-GTP causes cargo binding of export receptor. Ran-GTP causes cargo release in the nucleus and GTP-bound receptors return to cytosol.

Nuclear localization of activated T cells control gene expression

Nuclear lamina Is a meshwork of interconnected protein subunits called nuclear lamins. → Lamins are special class of intermediate filament proteins that polymerize into a two-dimensional lattice. Gives shape and stability to the nuclear envelope, interact with nuclear pore complexes, integral proteins and chromatin.

The breakdown and reformation of the nuclear envelope during mitosis Phosphorylation of the lamins is thought to trigger the disassembly of the nuclear lamina, which in turn causes the nuclear envelope to break up. Dephosphorylation of the lamins is thought to thelp to reverse the process. NLS is not cleaved off after transport into nucleus.

The transport of proteins into mitochondria and chloroplasts In contrast to the cristae of mitochondria, the thylakoids of chloroplasts are not connected to the inner membrane and therefore form a compartment with a separate internal space. Mainly depending on the import of proteins from the cytosol. → proteins need to be translocated.

Translocation into mitochondrial matrix depends on a signal sequence and protein translocators at N terminus, rapidly removed after import by a protease (signal peptidase) in matrix. folded into an amphipathic α helix, positively charged (red) clustered on one side of the helix while uncharged hydrophobic (yellow) are clustered primarily on the opposite face.

Protein translocators TOM complex: translocase for outer membrane TIM complex: translocase for inner membrane Both complexes contain components that act as receptors for mitochondrial precursor proteins and other components that form the translocation channel. OXA complex: protein translocator in the inner mitochondrial membrane. mediates the insertion of inner membrane proteins that synthesized within the mitochondria. helps to insert some protein that are initially transported into the matrix by TOM and TIM complexes.

Protein import by mitochondria TOM complex first transports the mitochondrial targeting signal across the outer membrane. Reaches in the intermembrane space, targeting signals binds to a TIM complex, opening the channel in the complex through which the polypeptide chain either enters the matrix. The signal sequence is cleaved off by a signal peptidase in the matrix to form the mature protein. The free signal sequence is then rapidly degraded.

ATP hydrolysis and a H+ gradient are used to drive protein import into mitochondria (1) Bound cytosolic hsp70 is released from the protein in a step that depends on ATP hydrolysis. After the initial insertion of the signal sequence and of adjacent portions of the polypeptide chain into TOM complex, the signal sequence interacts with a TIM complex. (2) The signal sequence is then translocated into the matrix in a process that requires

Two plausible models of how mitochondrial hsp70 could drive protein import In the thermal ratchet model, the translocating polypeptide chain slides back and forth, driven by thermal motion, and it is successively trapped in the matrix by hsp70 binding. In both models, hsp70 binds to the TIM23 complex, which loads the hsp70 onto the translocating polypeptide chain as it emerges from the complex into the matrix. In the cross-bridge ratchet model, a conformational change in hsp70 actively pulls the chain into the matrix.

Protein import from the cytosol into the inner mitochondrial membrane or intermembrane space

Translocation of a precursor protein into thylakoid space of chloroplasts Two signal sequences are required for proteins directed to thylakoid membrane in chloroplasts. Signal sequences for mitochondria and chloroplasts are diffferent. Four routes of translocation into thylakoid membrane

Peroxisomes Contain high concentrations of oxidative enzymes (catalase and urate oxidase). Major sites of oxygen utilization. Use molecular oxygen to remove hydrogen atoms from specific organic substrates (R) in an oxidative reaction that produces hydrogen peroxide. RH2 + O2 → R + H2O2 Catalase utilizes H2O2 Surrounded by a single membrane and do not contain DNA or ribosomes. Acquire their proteins by selective import from the cytosol.

A model for how new peroxisomes are produced A short signal sequence (3 amino acids located at the C-terminus or near N-terminus) directs the import proteins into peroxisomes. Peroxisomes are thought to form only form preexisting peroxisomes by a process of growth and fission.

Endoplasmic reticulum ER membrane separates the ER lumen from cytosol and mediates the selective transfer of molecules between these two compartments. play central role in lipid and protein biosynthesis. captures proteins from the cytosol i) transmembrane proteins which are partly translocated across the ER membrane and become embeddedin it. ii) water-soluble proteins which are fully translocated across the ER membrane and are released into the ER lumen. Ribosome is directly attached to the ER membrane. → synthesizing protein. Two spatially separate populations of ribosomes in the cytosol: i) membrane-bound ribosomes ii) free ribosomes

Free and membrane-bound ribosomes A common pool of ribosomes is used to synthesize the proteins that stay in the cytosol and those that are transported into the ER. The ER signal sequence on a newly formed polypeptide chain directs the engaged ribosome to the ER membrane. The mRNA molecule remains permanently bound to the ER as part of a polyribosome, while the ribosomes that move along it are recycled. At the end of each round of protein synthesis, the ribosomal subunits are released and rejoin the common pool in the cytosol.

The signal hypothesis: protein translocation across the ER membrane When the ER signal sequence emerges from the ribosome, it directs the ribosome to a translocator on the ER membrane that forms a pore in the membrane through which the polypeptide is translocated. The signal sequence is clipped off during translation by a signal peptidase, and the mature protein is released into the lumen of the ER immediately after being synthesized.

The signal-recognition particle (SRP) Mammalian SRP is an elongated complex containing six subunits and one RNA molecule (SRP RNA). One end of the SRP binds to an ER signal sequence on a growing polypeptide chain, while the other binds to the ribosome itself and pauses translation. The RNA in the particle may mediate an interaction with ribosomal RNA. The crystal structure of the signal-sequence-binding domain of a bacterial SRP subunit. The domain contains a large, exposed binding pocket that is lined by hydrophobic amino acids, a large number of which are methionines. The outline of the pocket is shaded in gray to emphasize its location. The flexible side chains of methionine are ideal for building adaptable hydrophobic binding sites for other proteins.

How ER signal sequences and SRP direct ribosomes to the ER membrane The SRP binds to both the exposed ER signal sequence and the ribosome, thereby inducing a pause in translation. The SRP receptor in the ER membrane, which it is composed of two different polypeptide chains, binds the SRP-ribosome complex and directs it to the translocator. The SRP and SRP receptor are then released, leaving the ribosome bound to the translocator in the ER membrane. The translocator then inserts the polypeptide chain into the membrane and transfers it across the lipid bilayer. SRP release occurs only after the ribosome has become properly engaged with the translocator in the ER membrane. The translocator is closed until the ribosome has bound, so that the permeability barrier of the ER membrane is maintained at all times.

Three ways in which protein translocation can be driven through structurally similar translocators

A model for how a soluble protein is translocated across ER membrane Upon binding an ER signal sequence, the translocator opens its pore, allowing the transfer of the polypeptide chain across the lipid bilayer as a loop. After the protein has been completely translocated, the pore closes, but the translocator now opens laterally within the lipid bilayer, allowing the hydrophobic signal sequence to diffuse into the bilayer, where it is rapidly degraded.

How a single-pass transmembrane protein with a cleaved ER signal sequence is integrated into the ER membrane Co-translational translocation process is initiated by an N-terminal ER sequence that functions as a start-transfer signal. In addition to this start-transfer, the protein also contains a stop-transfer sequence. When the stop-transfer sequence enters the translocator and interacts with a binding site, the translocator changes its conformation and discharges the protien laterally into the lipid bilayer.

Protein glycosylation in the rough ER Almost as soon as a polypeptide chain enters the ER lumen, it is glycosylated on target asparagine amino acids. The precursor oligosaccharide is transferred to the asparagine as an intact unit in a reaction catalyzed by a membrane-bound oligosaccharyl transferase enzyme. One copy of this enzyme is associated with each protein translocator in the ER membrane.

The role of N-linked glycosylation in ER protein folding The ER-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the ER. Removal of the terminal glucose by a glucosidase releases the protein from calnexin. A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not. If the protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the N-linked oligosaccharide, renewing the protein’s affinity for calnexin and retaining it in the ER. The cycle repeats until the protein has folded completely.

The export and degradation of misfolded ER proteins Misfolded soluble proteins in the ER lumen are translocated back into the cytosol, where they are deglycosylated, ubiquitylated and degraded in proteasomes. Misfolded membrane proteins follow a similar pathway. Misfolded proteins are exported through the same type of translocator that mediated their import; accessory proteins that are associated with the tranlocator allow it to operate in the export direction.

The unfolded protein response in yeast

Phospholipid exchange proteins help to tranport phospholipids from the ER to mitochondria and peroxisomes Phospholipids are insoluble in water, their passage between membranes requires carrier proteins. Phospholipid exchange proteins are water-soluble proteins that carry a single molecule of phospholipid at a time. They can pick up a lipid molecule from one membrane and release it at another, thereby redistributing phospholipids between membrane-enclosed compartments. The net transfer of phosphatodycholine (PC) from the ER to mitochondria can occur without the input of additional energy because the concentration of PC is high in the ER membrane and low in the outer mitochondrial membrane.