- List the benefits of intracellular organelles to eukaryotic cells.
- Describe how microtubules and motors are used to position organelles in the cytoplasm of cells.
- Define the role of signal sequences in targeting proteins to different organelles.
- Contrast protein import into the ER and peroxisomes.
Eukaryotic cells contain collections of proteins that function as a unit called organelles. Some of these organelles are surrounded by a membrane similar in structure to the cell membrane but with a different composition of protein and phospholipid.
Membrane-bound organelles offer several advantages to eukaryotic cells. First, cells can concentrate and isolate enzymes and reactants in a smaller volume, thereby increasing the rate and efficiency of chemical reactions. Second, cells can confine potentially harmful proteins and molecules in membrane-bound organelles, protecting the rest of the cells from their harmful effects. For example, the lysosome, which is a membrane-bound organelle, contains many enzymes that digest protein, nucleic acids and lipids. If these enzymes were released in the cytosol, they could chew up the cell's proteins, nucleic acids and lipids, leading to cell death. The membrane surrounding the lysosome keeps those digestive enzymes away from the rest of the cell.
Microtubule Organization of Cytoplasm
Organelles and proteins are usually not randomly distributed throughout the cell but are organized by localizing them to regions where they are needed. The cell utilizes microtubules and motor proteins to help localize organelles. Microtubules are long filaments that extend throughout the cytoplasm. Two types of motor proteins, kinesins and dyneins, walk along microtubules and generate force to pull organelles through the cytoplasm.
Microtubules are polymers of a heterodimer of alpha and beta tubullin. Tubulin polymerizes into linear protofilaments and a microtubule contains 13 protofilaments arranged in a cylinder with a hollow core. Microtubules are polarized into a minus end and plus end. Microtubules grow from their plus ends by adding more tubulin subunits. The minus ends of microtubules are unstable and are stabilized by proteins in the microtubule organizing center (MTOC). If the MTOC is in the center of a cell, microtubules radiate outward with their plus ends toward the plasma membrane
Kinesins and dyneins walk along microtubules by utilizing the energy from ATP hydrolysis. Both sets of proteins contain motor domains that bind microtubules and hydrolyze ATP. The motor domains generate movement along microtubules. Most kinesins walk toward the plus end of microtubules, whereas dynein walks toward the minus end. This gives cells two tools to control the distribution of organelles along microtubules. Kinesins and dyneins also contain a cargo-binding domain that links them to different organelles. Kinesins are a large family of proteins and the cargo binding domain is the most divergent, allowing different members of the kinesin family to bind different organelles. Dynein is a large complex of several proteins and how it binds cargo is less clear.
Actin filaments also support the transport of cellular material but over much shorter distances than microtubules. Actin filaments are a polymer of actin which is a small globular protein. The actin filament is a helical array of actin and similar to microtubules has a plus and minus end with filaments growing more readily from their plus ends. Actin filaments lack the extensive lateral contacts of microtubules and usually are much shorter than microtubules. Actin filaments tend to localize near the cell membrane where they provide structural support.
Myosins are a class of motor proteins that can generate force along actin filaments. Some myosins are involved in cell contraction (i.e. contraction of muscle), whereas others support the movement and positioning of organelles. Class V myosins are involved in the transport of organelles in several different types of cells. Similar to the structure of kinesin, class V myosins contain a motor domain that binds actin filaments and use the energy of ATP hydrolysis to walk along filaments. The C-terminus of myosin V binds organelles.
To transport and position organelles, cells often use both microtubules and actin filaments. Microtubules, kinesins and dyneins are used to move organelles over long distances (several microns or more), whereas actin filaments transport organelles over short distances (e.g. near the plasma membrane). Often an organelle will contain more than one type of motor protein (e.g. kinesin and myosin V) to allow cells to utilize both sets of filaments to position the organelle.
To maintain the identity and function of the different organelles and plasma membrane, cells need to target specific proteins to organelles and other intracellular compartments. Most of these proteins contain a short sequence, called a signal sequence, that determines their intracellular location. Signal sequences can be localized anywhere in a protein but are often found in the N-terminus. Signal sequences that target proteins to the same organelle often do not share the same primary sequence. It is usually the overall biochemical properties of the sequence that determine whether it targets a proteins to an organelle. Signal sequences are used to import both soluble proteins and integral membrane proteins.
Importing proteins into membrane-bound organelles
Because the membranes that surrounds organelles restricts the passage of proteins, organelles have evolved different mechanisms for importing proteins from the cytoplasm. Most organelles contain a set of membrane proteins that form a pore. This pore allows the passage of proteins with the correct signal sequence. Some pores (ER, mitochondria) can only accommodate unfolded proteins, whereas other pores (nucleus, peroxisome) allow folded proteins to pass.
Targeting Proteins to the Endoplasmic Reticulum
Proteins destined for secretion, the plasma membrane or any organelle of the secretory pathway are first inserted into the ER. Most proteins cross the ER co-translationally, being synthesized by ribosomes on the ER. Both soluble proteins (proteins that reside in the lumen of organelles or are secreted) and integral membrane proteins are targeted to the ER and translocated by the same mechanism.
The signal sequence for ER proteins usually resides at the N-terminus. The signal recognition particle (SRP), a complex of 6 proteins and one RNA, binds the signal sequence immediately after it is translated. The SRP also interacts with the ribosome and stops translation. The surface of ER membranes contains a receptor for SRP. The SRP receptor recruits SRP, nascent ER protein, and ribosome to the ER. The SRP receptor releases the SRP from the signal sequence and allows translation to continue on the ER membrane.
Ribosomes on the ER membrane bind to the protein translocator. The translocator is a transmembrane protein that forms a aqueous pore. The pore is the channel through which the newly synthesized ER proteins will be translocated across the ER membrane. Translation of the ER protein generates the “force” to push the ER protein through the channel.
Soluble proteins are completely translocated through the channel; the signal sequence remains in the channel and is cleaved from the rest of the protein by a protease in the lumen of the ER.
Integral membrane proteins contain a stop transfer sequence downstream from the signal sequence. The stop transfer sequence ceases translocation through channel and the portion of the protein after the stop transfer sequence resides outside the ER. Integral membrane proteins can be translocated such that either their N-terminus or C-terminus resides in the lumen of the ER. Proteins with their C-terminus in the lumen tend to have an internal signal sequence. The translocator appears to open on one side to allow integral membrane proteins to diffuse into the surrounding lipid bilayer.
Some proteins span the membrane several times and these proteins contain after the stop transfer sequence a start transfer sequence that reinitiates translocation of the protein through the channel. A protein with a signal sequence, stop transfer and start transfer would span the membrane twice with a loop residing in the cytosol or lumen. To generate proteins that span the membrane several times, the protein would need several alternating stop and star transfer sequences.
Once proteins enter the ER, they fold into their three dimensional structures. Several mechanisms exist to help fold proteins, including chaperones and glycosylation. The ER also contains mechanisms to handle proteins that fail to fold.
Targeting Proteins to Mitochondria
Although mitochondria contain their own genome, most mitochondrial proteins are encoded by nuclear genes, necessitating a mechanism to target and import those proteins into mitochondria. Similar to proteins imported into the ER, mitochondrial proteins contain a signal sequence that targets them to mitochondria. Unlike ER proteins, mitochondrial proteins are imported post-translationally. Because proteins must be unfolded to translocate through channels in the mitochondrial membrane, mitochondrial proteins are kept unfolded in the cytosol by chaperones.
Protein import into mitochondria is similar to import into the ER but is complicated by the presence of two membrane around mitochondria. Mitochondrial proteins can reside in the outer membrane, inner membrane, intermembrane space, or matrix (space inside inner membrane).Thus, mitochondria have translocators that allow passage of proteins across the outer membrane and across the inner membrane. The TOM complex mediates passage across the outer membrane whereas the TIM complex mediates passage across the inner membrane.
Translocation of Proteins into Mitochondria
The signal sequence that targets proteins to the matrix usually resides at the N-terminus. The signal sequence is recognized by proteins in the TOM complex. The TOM complex passes the proteins into the inner membrane space where the TIM complex in the inner membrane passes the protein into the matrix. The TOM and TIM complex often work together to translocate a protein across both membranes. Translocation across mitochondrial membranes is energy dependent. Chaperones within the matrix help “pull” the protein across the inner membrane and require ATP hydrolysis to function. The proteins fold inside the matrix.
Proteins targeted to the inner membrane use a similar mechanism as matrix proteins but contain a stop transfer sequence recognized by the TIM complex. Proteins targeted to the outer membrane are translocated across the outer membrane into the intermembrane space and then imported into the outer membrane by the SAM translocator. Proteins targeted for the intermembrane space are partially inserted into the inner membrane and then cleaved by a protease and released into the inner membrane space.
Import and Export of Nuclear Proteins
In contrast to the ER and mitochondria, the nucleus imports primarily soluble proteins. In addition, proteins often shuttle between the nucleus and cytoplasm and the cell uses nuclear import/export to regulate several critical biochemical pathways. The nucleus is surrounded by two membranes and embedded in these membranes are thousands of nuclear pores through which proteins and other macromolecules (RNA, ribsosomes) enter and exit the nucleus. Nuclear pores are stabilized in membranes by lamins, a cytoskeletal network that underlies the inner nuclear membrane and provides structural support to the membrane. The nuclear pore restricts passage of material based on size: things smaller than ~ 30 kD freely diffuse across the pore but large molecules need a way to get in and out. Proteins that traffic into the nucleus contain a nuclear import signal and those that must also exit the nucleus contain a nuclear export sequence.
Distinguishing Cytoplasm from Nucleoplasm
To generate directed transport of proteins into and out of the nucleus, proteins must know whether they are in the cytoplasm or inside the nucleus. To differentiate between the nucleus and cytoplasm, cells use a small GTP-binding protein called Ran. Like all GTP-binding proteins, Ran exists either in a GTP-bound state or GDP-bound state. Two proteins catalyze the switch between these states. Ran-GAP (GTPase activating protein) catalyzes GTP hydrolysis generating Ran-GDP. Ran-GEF (guanine nucleotide exchange factor) catalyzes release of GDP and rebinding of GTP, generating Ran-GTP. Ran-GAP localizes to the cytoplasmic side of nuclear pores whereas Ran-GEF associates with chromatin and therefore localizes to the nucleus. As a result, most Ran in the nucleus is bound to GTP and most Ran in the cytoplasm is bound to GDP.
Receptors (importins) bind nuclear import sequences in proteins. Importins also interact with filaments that extend off the cytoplasmic side of nuclear pores. By an unknown mechanism, importins bound to their cargo traffic through the nuclear pore. Inside the pore the importin-cargo complex encounters Ran-GTP. Ran-GTP dissociates importins from the cargo, releasing cargo proteins to do their work in the nucleus.
Many proteins that enter the nucleus must be exported to the cytoplasm (e.g. importins). These proteins contain a nuclear export sequence that interacts with a receptor called exportin. Ran-GTP binds to this exportin-cargo complex and stabilizes the interaction. The exportin-cargo-RanGTP complex traffics through the pore (mechanism unclear) where it encounters Ran-GAP on the cytoplasmic side. Ran-GAP converts Ran-GTP to Ran-GDP causing exportin to dissociate from its cargo.
Importing proteins into peroxisomes and Zelleweger's Syndrome
Peroxisomes are small organelles (~ 1 µm in diameter) that perform a variety of functions for cells. Peroxisomes metabolize harmful chemicals (phenols, formaldehyde, ethanol), metabolize fatty acids, and catalyze a step in the synthesis of plasmalogen which is a lipid found in myelin.
Proteins targeted for peroxisomes contain a signal sequence that is recognized by a family of proteins called Pex proteins. Some of these Pex proteins bind to signal sequences whereas other for a pore in the membrane of peroxisomes that allows the entry of peroxisome proteins.
Cells that contain mutations in Pex proteins cannot import proteins into peroxisomes and consequently, these cells lack peroxisomes. Mutations in Pex proteins are associated with a set of diseases called Zelleweger's Syndrome. In Zelleweger's Syndrome, infants lack muscle tone and often the ability to suckle. Infants also display craniofacial abnormalities and an enlarged liver. The prognosis for infants suffering from Zelleweger's Syndrome is poor with most not surviving beyond a year.
Because peroxisomes contribute to the synthesis of a lipid found in myelin, patients with Zelleweger's often display poor myelination of neurons. Myelination is critical for the function of neurons in conducting signals to target cells.