Class Note for BME 510 at UA
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Date Created: 02/06/15
BME 510 Lynch Lecture 3 October 27 2008 Protein Sorting and Regulated Secretion Reading Lodish CH 14 Review of Cell Architecture r39u V39brnsnu H L wuclenus l llleSClmEi l llllr lL39WLl 42le Ly 309lrlu Colosgl Pathway for Protein Production and Sorting Translation of mRNA occurs in the cell cytosol The mRNA quotdiffusesquot from the nucleus and associates With Ribosomes The nal location of the ribosome mRNA complex may be dictated by signals exposed on the RNA or by the complex itself The best example of directed localization of Ribosome mRNA occurs during translation of proteins destined for the plasma membrane or for secretion Studies of this mechanism have led to the determination of speci c signal sequences expressed on proteins which direct their movements to speci c compartments Within cells RVBOSOMES retention ralentlan Diagam oi biosynthetic protein trailic Signals that direct a protein s movennent through the systenn are CWOSO L contained in its amino acid sequence At each intermediate location compartment a decision is ENDDPLASMIC HETICULUM rmntnon made 3510 whether to retain the protein 0quot MITOCHONDRIA PERCXISOMES GO to move iunther along the pathway In principal signals can be required ior either retention in or ior J SECRETORY leaving each compartment shown with the alternative UCLEUS iate being a deiault pathway that requires no signal CELL SURFACE BME 510 Lynch Lecture 3 October 27 2008 Sorting and the Golgi Apparatus Regulated and Constitutive Secretion The regulated pathway for secretion of protein requires the presence of internal signal peptides that guide the protein to vesicles for storage While in the storage vesicles the proteins are processed from prehormones to active peptides The vesicles are mobilized for secretion only after processing is complete and a cellular second messenger signals secretory stimuli On the other hand the constitutive pathway requires no stimuli rather this pathway is continuous All trans membrane proteins and extracellular matrix components are secreted continuously through the constitutive pathway Secretion or expression of these proteins at the cell surface is regulated at the level of transcription andor translation only The absence of any secondary signal guarantees transport quickly to the cell surface Moreover any proteins that are missorted into the ER will not have retention signals and are therefore ushed from the cell via this constitutive pathway Sorting to the Lvsosomal and Endocvtic Compartments The endocytic pathway refers to removal of membrane from the cell surface and incorporation into intracellular membrane compartments the nature of which are not completely understood During the process of membrane retrieval specific membrane proteins are internalized and substances outside the cell but near the surface maybe receptor associated can be internalized pinocytosis The contents of the membrane and vesicle can be moved to lysosomal compartments where acid hydrolases degrade most nonlipids This can include modification of proteins such as receptors which can be cycled back to the plasma membrane In epithelial cells movement from one surface to the other is referred to as transcytosis but is likely to process in the same compartments only with unique signals to direct the contents to either the apical or basolateral membranes Sorting of acid hydrolases from the Golgi to lysosomes has been described in detail Acid hydrolases are glycosylated in the ERGolgi then in the trans golgi Mannose6Phosphate M6P groups are added Only peptides with M6P markers are incorporated into vesicles destined to be lysosomes and this is directed by a receptor protein for the M6P As indicated by their names these hydrolases are most active under acidic conditions pHlt5 Since vesicular pH is near 7 initially vesicle pH must decreases A H ATPase Vtype is responsible for the acidification but this protein is not tagged by M6P Therefore the Vtype ATPase may come from an endocytic route and elicit acidification only after endocytic substances become incorporated The acid hydrolyses remain membrane attached via the receptor until acidification occurs then the receptors are cycled back to the golgi Thus it appears that all intacellular compartments can be in contact with the exception of mitochondria A current argument proposes vesicles observed by microscopy are actually part of a continuous cellular membrane compartment through which substances travel in specific directions due to intramolecular signals Elimination of Missorted Proteins i U biquination as a signal for protein degradation Signals on peptides also exist to protect them from or promote degradation Specific amino acids at the immediate N terminus act as signals although other signals are believed to exist Most cytosolic proteins have stabilizing amino acids at their Cteminus whereas those destined for the ER do not In this way a protein that is translated improperly or missorted is rapidly degraded A multi enzyme complex recognizes the de stabilizing amino acid and convalently links a small peptide call ubiquitin to the missorted protein After multiple ubiquitins are added on top of one another this ubiquinated protein is marked for rapid destruction ii Constitutive Secretion Any protein in the ER that has no additional targeting domain is secreted from the cell via the constitutive pathway BME 510 Lynch Lecture 3 October 27 2008 Regulated Secretion Regulation of pH in Intracellular Compartments The regulation of pH within subcellular compartments is crucial for maintaining macromolecular traf cking from one intracellular compartment to another In the endocytic pathway receptorbound ligands and dissolved substances in the extracellular uid are taken up at the plasma membrane by specialized structures that form early endosomes The endosomal lumen rapidly acidifies inducing the dissociation of endocytosed receptorligand complexes The endosomal contents may be recycled back to the plasma membrane as in the transferrin system or undergo further degradation in late endosomes and lysosomes In the regulated exocytic pathway of hormone secreting cells the acidic environment of the secretory vesicle regulates the proteolytic processing of prohormones into the mature form of the secreted peptide For example in the pancreatic beta cell the cleavage of the proinsulin B chain and C chain by a specific endopeptidase requires an acidic environment within the secretory granule Conversely it has been suggested that alkalinization of the secretory granule lumen upon activation of secretion should occur to promote solubility of the stored and condensed insulin to enhance its release from the granule upon fusion with the plasma membrane Regulated Secretion Regulated secretion takes place in all endocrine cell types and general rules hold for all these cells For example maturation of secretory granules SG requires lumenal acidi cation with activation of processing enzymes The subsequent processing depends on the speci c peptide that needs to be processed so maturation time is variable between cells type In addition only a fraction of the total compliment of SG within a given cell are in a secretable form this again varies for cell type with anywhere from 10 beta cell to 50 adrenal chromaf n cell in secretable form Another commonality is the second messenger pathways for activation of secretion In nearly all secretory cell types elevations in intracellular Ca initiate secretion and elevation in cAMP levels potentiate this activated response but do not directly activate secretion Finally there appears to be a large degree of homology in the proteins used to target the SG to the membrane for release SNARE s NSF Attachment Protein Receptor SNARE s are complexes of proteins which form at the plasma membrane between vesicle surface proteins and proteins on the inner lea et of the plasma membrane The number of proteins in these complexes can vary but the usual suspects are NSF SNAP Synaptosome Associated Protein syntaxins synaptobrevin orVAMP and synaptotagmin As the names suggest many were initially isolated from synapses but homologs for many of these proteins have subsequently been isolated from nonneuronal endocrine tissues as well tSNARE s are the proteins found on the target membrane which for exocytosis is the plasma memebrane e g syntaxinl and vSNARE s are those on the vesicular membranes e g VAMP Synaptophysin and Synaptotagmin Both proteins were rst isolated from neuronal tissues synaptosomes but are now known to be widely expressed in neurons and endocrine cells Synaptophysin is the most abundant protein in synaptic vesicle membranes 7 of total synaptic vesicle protein This protein likely organizes the vesicle membrane and may act as a pore for release of vesicle contents upon insertion into the plasma membrane Synaptotagmin is the primary candidate for the secretory granule Ca2 sensor For example it s been shown in cell extracts that increasing Ca2 promotes SNARE formation only in the presence of synaptotagmin Antibodies to both proteins are commercially available and therefore are used routinely to identify secretory granule distributions within isolated cells Synaptotagmin also interacts with the receptor for black widow venom Olatrotoxin which resides in the synaptic plasma membrane Binding of the toxin leads to massive exocytosis of synaptic vesicles Work using this toxin suggests that both the O latrotoxin receptor and synaptotagmin are part of a secretory granule fusion complex at the docking site of sysnaptic vesicles at the presynaptic membrane BME 510 Lynch Lecture 3 October 27 2008 Potentiation of Secretion In many secretory cell types elevation of cAMP and in some cells cGMP will potentiate the release of secretory granule contents during activated secretion Potentiation occurs when two second mssenger pathways are simultaneously activated and acting on two different mechanisms drive the same process in this case secrtion The result of this duality of activation is a greatly enhanced secretion more than the additive effect of either activator alone The mechanism by which these second messengers act is not completely understood Clearly cAMP activation of protein kinase A is required but the basis for the subsequent actions of PKA are not known Similarly cGMP likely affects protein phosphatase activities in these cell types but again the specific mechanisms though which this process acts to modulate secretion is unknown Since synapsin phosphorylation may mediate binding and synaptic vesicles to the cytoskeleton this mechanism has been suggested to be involved in mobilization of secretory granules to the plasma membrane However there is direct evidence for this process In addition to date synapsin homologs in nonneuronal tissue have not been identified Alternatively alterations in secretory granule ion contents may modify the storage state of the hormonenuerotransmitter within the secretory granule prior to insertion of the secretory granule into the plasma membrane Such changes in secretory protein configuration may facilitate their release References Burgoyne RD and G Alvarez de Toledo 2000 Fusion Proteins and Fusion Pores Regulated exocytosis and vesicle cycling EMBO Reports 11 301307 Tompkins LS SM Murphy KD Nullmeyer C Weber and RM Lynch 2002 Regulation of secretory granule pH in insulin secreting cell lines Am J Physiol Cell Physiol 2832C42937
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