1. to understand how proteins find their destination in prokaryotic and eukaryotic cells 2. to know how proteins are bio-recycled

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1 Protein Targeting Objectives 1. to understand how proteins find their destination in prokaryotic and eukaryotic cells 2. to know how proteins are bio-recycled As a protein is being synthesized, decisions must be taken about sending it to the correct location in the cell where it will be required. The information for doing this resides in the nascent protein sequence itself. Once the protein has reached its final destination, this information may be removed by proteolytic processing. Targeting in Bacteria In bacterial cells, the targeting decision is relatively straightforward: is the protein destined to be an intracellular protein or an extracellular one? 1

2 Secreted proteins contain a signal sequence. This is a short (6-30) stretch of hydrophobic amino acids, flanked on the N-terminal side by one or more positively charged amino acids such as lysine or arginine, and containing neutral amino acids with short sidechains (such as glycine or alanine) at the cleavage site. As proteins with signal sequences are synthesized, they are bound by the SecB protein. This prevents the protein from folding. SecB delivers the protein to the cell membrane where is secreted through a pore formed by the SecE and SecY proteins. Secretion is driven by the SecA ATPase. After the protein has been secreted, the signal sequence is removed by a membrane bound leader peptidase. 2

3 Targeting in Eukaryotes In eukaryotic cells, the situation is more complex. Extracellular proteins can be targeted for secretion or to the cell membrane, or to one of the many internal organelles such as the lysosome. Intracellular proteins can be targeted for the cytoplasm, to the nucleus or to special organelles such as the mitochondrion. The Signal Sequence hypothesis was first enunciated by Gunther Blöbel who was awarded the Nobel Prize in Medicine in 1999 for his work. The following diagram summarizes the choices/fates available to newly synthesized proteins in a eukaryotic cell: 3

4 Protein secretion in eukaryotic cells also involves a signal sequence. As in bacteria, the signal sequence is a short (6-30) stretch of hydrophobic amino acids, flanked on the N-terminal side by one or more positively charged amino acids such as lysine or arginine, and containing neutral amino acids with short side-chains (such as glycine or alanine) at the cleavage site: The SRP cycle The signal recognition particle (SRP) associates with ribosomes that are in the process of translating the mrna for a secretory protein. The protein has a signal at the N-terminus. Subsequently, the ribosome-bound SRP interacts with the SRP-receptor a component of the ER membrane. Finally, SRP recycles to associate with another ribosome, and translation continues with the secretory protein transversing the membrane through a channel called the translocon. 4

5 Proteins that must be targeted to the nucleus have a nuclear localization signal (NLS). Once common type of signal is a series of five or so closely spaced positively charged amino acids. Protein Turnover Protein lifetimes must also be regulated. Some proteins are needed for only very short times -- and could be harmful is present for too long. Others are needed all the time and it would be unnecessarily wasteful to keep re-synthesizing them. The following table, demonstrates the great differences in lifetimes of some rat liver enzymes: 5

6 Enzyme Half-life (h) Ornithine decarboxylase 0.2 RNA polymerase I 1.3 Tyrosine aminotransferase 2.0 Serine dehydratase 4.0 PEP carboxylase 5.0 Aldolase 118 GAPDH 130 cytochrome c 150 The lifetime of proteins in eukaryotes appears to be determined by the nature of the N-terminal amino acid. Some amino acids (e.g. Ala, Cys, Gly, Met, Pro, Ser, Thr, Val) stabilize proteins (at least in yeast); others (e.g. Arg, His, Ile, Leu, Lys, Phe, Trp, Tyr) destabilize proteins. 6

7 Targeted Protein Degradation In order to keep a cell working it needs to remove: 1. incorrectly synthesized proteins (with errors in amino acid sequence) 2. damaged proteins (i.e. oxidative damage) 3. cell-cycle specific proteins 4. other signaling proteins which are no longer necessary One mechanism of protein degradation is via lysosomes. Lysosomes are acidic vesicles that contain about 50 different enzymes involved in degradation: 1. proteases (cathepsins): cleave peptide bonds 2. phosphatases: remove covalently bound phosphates 3. nucleases: cleave DNA/RNA 4. lipases: cleave lipid molecules 5. carbohydrate-cleaving enzymes: remove covalently bound sugars from glycoproteins Lysosomes often secrete their contents into the extracellular medium via exocytosis. Lysosomes can also target damaged organelles in a process called autophagy. Sometimes, lysosomes are triggered to rupture inside a cell, resulting in autolysis, also called apoptosis or programmed cell death. 7

8 Another major mechanism is via ubiquitin labeling of surplus proteins: Ubiquitin (a small 76-residue protein) is attached to the protein: o o o First, an activating enzyme attaches itself to the carboxy terminus of free ubiquitin in an ATP-dependent process. Then, the activated ubiquitin is transferred onto a second enzyme which at the same time recognizes damaged proteins. The activated ubiquitin is then covalently linked to lysine residues on the surface of the damaged protein. These ubiquitin-tagged proteins are now recognized by specific proteases in the cytosol which in turn cleave and degrade the tagged protein. These proteases are combined in a very large protein complex called the proteasome. 8

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10 Conclusions: 1. Proteins synthesized on cytosolic ribosomes are released into the cytosol or transported into mitochondria, peroxisomes, and nucleus. Proteins synthesized on ribosomes attached to the rough endoplasmic reticulum (RER) are destined for lysosomes, cell membranes, or secretion from the cell. These proteins are transferred to the Golgi complex, where they are modified and targeted to their ultimate locations. 2. In order to keep a cell working it needs to remove unwanted protein 11