weber uiuc edu 02 November 2007 MCB150 Lecture 29 Lecture 29 02 November 2007 Movie Time This is the second movie that wasn t shown at the end of Monday s lecture This shows how crowded the cell is It isn t that unusual that things find where they need to go They aren t dependent upon random interactions However things are pretty close to each other so it isn t that strange they can bump into each other occasionally Lysosomes Still on the subject of how proteins end up where they are supposed to be Still kind of in the secretory pathway though now we are in a side branch Lysosomes are the garbage collectors of the cell The cell uses lysosomes to chop up things and recycle them MICROGRAPH Here we see some densely staining regions These are lysosomes o They are irregularly shaped because they have a variety of materials within them o They are trash compactors and are working on whatever they have to degrade o They stain densely because they are packed with garbage and enzymes The other big objects are mitochondria o They are similarly sized Gives an idea of scale Lysosomal Acid Hydrolysis How do cells get rid of macromolecules from the extracellular space or from an object targeted for removal within the cell The enzymes that do the job are lysosomal acid hydrolases Lysosomal acid hydrolases break down macromolecules to recycle the parts o Lipids proteins NAs polysaccharides etc Need to have enzymes to degrade each of these o Proteins proteases o Nucleic acids nucleases o Lipids lipases o Polysaccharaides glycosidases Lots of different versions of each of these enzymes They come together into a toolbox There are about 50 different kinds of enzymes Not just 50 enzymes total Need a set of every one of the 50 Called acid hydrolases for a reason they are optimally active at an acidic pH 5 o This is important to know Why 1 Sequestration keep them locked up 2 Acid hydrolases only active at acidic pH o These things keep the hydrolases from operating on the cell s contents Lysosomes are compartments away from the rest of the cell The hydrolases require acidic pH so if the lysosome bursts the enzymes are inactivated in the more basic neutral pH 7 2 of the cytosol o That is the cytosol buffers the rupture Maintaining the lower pH requires a proton pump o This isn t a mitochondria Page 1 of 6 weber uiuc edu 02 November 2007 MCB150 Lecture 29 o This doesn t have an ETC to drive it in an energy efficient process Instead there is a proton pump It is an active transport mechanism It moves the charged molecules against the concentration gradient o Already more protons in the lumen of the lysosome compared to the cytoplasm and need to keep elevating it o It requires ATP ADP hydrolysis to drive it pumping protons against a concentration gradient o The increase of H lowers the pH o To keep the protons out of the rest of the cell the pump is a one way street Also protons being positively charged will not spontaneously cross the hydrophobic regions of the semi permeable bilayer The pump channels will keep the gate one way and will stay closed unless pumping in one direction o If you lost the ability to maintain the gradient then the enzymes would not be functional resulting in serious problems The lysosomes will eventually degrade themselves o The hydrolytic enzymes proteases will eventually degrade the protein coat on the interior of the lysosome and eventually lipidases will attack the lipid bilayer o The cell will make more lysosomes to compensate o The enzymes in the lysosome will be inactivated when it enters the cytosol buffer pH 7 2 The pump only works until the pH gradient is established after which it simply maintains the pH at 5 Lysosome Manufacture How are lysosomes rebuilt built It is a maturation process This usually starts with recognition of a molecule outside of the cell that needs to be degraded o It needs to be brought into the cell to be degraded o Perhaps the cell is responding to a signal o When the signal is no longer needed the signaling molecule needs to be brought in for degradation o E g a receptor that is causing a response Eventually that response is sufficient so you need to get rid of the signaling molecule Bring it in to destroy it o This process is called endocytosis DIAGRAM There are receptors on the primary membrane that receive the molecule that needs to be degraded These are green in the diagram o The blue molecules bind and now it is time to get rid of them o Pull it in and destroy the blue ones Invagination the process of budding off an endocytic vesicle from the cell membrane o The little endocytic vesicle will either run into other endocytic vesicles or into an endosome o The ones closest to the membrane are called early endosomes o These come in and fuse to make a new endosome or fuse with an existing endsome o Endsomes are under constant change There is always a process of bringing in new material for degradation Page 2 of 6 weber uiuc edu 02 November 2007 MCB150 Lecture 29 Once in the endosome the receptor lets go of the cargo molecule The cargo is an environment where things could start to be chopped up But the endosome is not a lysosome yet Why not o There are no acidic hydrolases o There is not an acidic environment In early endosomes you can find ATP dependent proton pumps in the membrane o A young endosome has a closer to neutral pH o The pumps will cause the pH to drop o Early endosomes are typically around pH 6 o We are just starting the process Not acidic enough just yet The receptor proteins need to be sent back to the membrane otherwise they will be degraded too Don t want to degrade these wasteful o The receptors will bud off Moving them back saves having to manufacture another receptor o They are recycled to the plasma membrane o The early endosome gives us the ability to recycle o The receptors are sequestered and sent back to the membrane in a transport vesicle o This process replenishes the lipid in the membrane and the membrane proteins avoiding the energy use of rebuilding everything o For MCB150 it is enough to know that this happens not necessarily how it happens Sequester and send them back The big blue chunks to be degraded will be sent to fuse with a late endosome Formation of an Active Lysomsome At this point there is an amorphous late endosome to which the early endosome will fuse The pH of a late endosome is now about 5 5 Still not optimal o Some hydrolases could begin working at this point o Enzyme activity is not ON OFF it is a