CHAPTER 12. Parts: ER, golgi complex, endosomes, lysosomes, vacuoles *Dynamic activities of endomembrane system are highly conserved

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1 CHAPTER 12 Overview f the Endmembrane System Parts: ER, glgi cmplex, endsmes, lyssmes, vacules *Dynamic activities f endmembrane system are highly cnserved - Materials are shuttled between rganelles frm the Glgi cmplex t the plasma membrane in a small, membranebunded transprt vesicles - Transprt vesicles: bud frm a dnr membrane cmpartment mve thrugh the cytplasm in a directed manner ften pulled by majr prteins that perate n tracks frmed by micrtubules and micrfilaments f the cytskeletn - Distinct pathways that have been studied - Bisynthetic pathway Prteins are synthesized in the ER, mdified during passage thrugh the glgi cmplex, and transprted frm the glgi t the varius destinatins (plasma membrane, lyssme, vacules) - Secretry pathway Many f the prteins synthesized in the ER are destined t be discharged (secreted) frm the cell 2 types Cnstitutive secretin Materials are transprted in secretry vesicles frm their sites f synthesis and discharged Int the extracellular space in a cntinual manner Engaged by mst cells Prcess that cntributes nt nly t the frmatin f the extracellular matrix but t the frmatin f the plasma membrane itself Regulated secretin Materials are stred as membranebund packages and discharged nly in respnse t an apprpriate stimulus (e.g. endcrine cells that release hrmnes; pancreatic acinar cells that release digestive enzymes, nerve cells that releases neurtransmitters) Materials t be secreted are stred in large, densely packed, membrane bund secretry granules Endcytic pathway Mvement f materials frm uter surface f cell t cmpartments such as endsmes and lyssmes lcated within the cytplasm Analgus t mvement f trucks carrying different types f carg alng the varius highways f the city Require defined traffic patterns t ensure materials are accurately delivered t the apprpriate sites Varius types f carg Secreted prteins Lyssmal enzymes * Membrane prteins are ruted t their apprpriate cellular destinatins by virtue f specific addresses r srting signals encded in the amin acid sequence f the prteins r in the attached ligsaccahrides * Srting signals are recgnized by specific receptrs that reside in the membranes r surface cats f budding vesicles (ensures that prtein is transprted t right destinatin) Mvement f the mlecules can be traced thrugh autradigraphy r thrugh Green Flurescent Prtein - Autradigraphy Use f radiactive materials Used t fllw the steps f a single cycle frm start t finish (frm the synthesis f a secretry prtein its discharge frm the cell) Prvides a means t visualize bichemical prcesses by allwing an investigatr t determine the lcatin f radiactively labeled materials within the cell Pulse-chase - Green Flurescent Prtein (GFP) Pulse: the brief incubatin with radiactivity f prtein during which labeled amin acids are incrprated int prtein Chase: perid when tissue is expsed t the unlabeled medium A perid during which additinal prteins are synthesized using nnradiactive amin acids Lnger the chase, farther the radiactive prteins manufactured during the pulse will have travelled frm their site f synthesis within the cell Utilizes a gene islated frm a jellyfish the encdes a small prtein which emits a green flurescent light Endplasmic Reticulum - 2 sub cmpartments: rugh ER (RER) and smth ER (SER) - Bth have a system f membranes that enclses a space, r lumen, that is separated frm the surrunding cytsl - Bth share many f the same prteins and engage in certain cmmn activities (synthesis f certain lipids and chlesterl) - RER - SER Typically cmpsed f a netwrk f flattened sacs (cisternae) Cntinuus with the uter membrane f the nuclear envelpe, which als bears ribsmes n its cystlic surface) Cntains prteins invlved in the mvement f nascent prteins int ER lumen The starting pint f bisynthetic pathway RER lumen is packed with mlecular chapernes that recgnize and bind t unflded r misflded prteins and give them the pprtunity t attain their crrect r native 3D structure Functins Site f prtein synthesis, carbhydrate chains and phsphlipids that jurney thrugh the membrane cmpartments Include secreted prteins, integral membrane prteins, sluble prteins that reside within cmpartments f endmembrane system Highly curved and tubular, frming an intercnnecting system if pipelines traversing the cytplasm Cntains reticulns:membrane-bending prteins which are largely absent frm flattened RER sheets; maintains the curvature f SER Functins: Synthesis f sterid hrmnes in the endcrine cells f the gnad and adrenal crtex Detxificatin in the liver (including barbiturates and ethanl, whse chrnic use can lead t prliferatin f the SER in liver Sequestering f calcium ins within the cytplasm f cells *The regulated release f calcium frm SER f skeletal and cardiac muscles cells ( sarcplasmic reticulumin muscle cells) triggers cntractin Plypeptides are synthesized at tw distinct lcales within the cell - One-third f prteins encded by mammalian genme are synthesized n ribsmes attached t cystlic surface f RER membranes Includes secreted prteins, intergral membrane prteins, sluble prteins - Other plypeptides are synthesized n free ribsmes(ribsmes nt attached t ER and subsequently released in cytsl Includes prtein destined t remain in cytsl, peripheral prteins f cystlic surface f membranes, prteins that are transprted t the nucles, perteins t be incrprated int perxismes, chlrplasts, and mitchndria

2 Determinatin f lcatin in a cell where a prtein is synthesized (signal hypthesis) - Site f synthesis f a prtein was determined by the sequence f amin acids in the N-terminal prtin f the plypeptide (first part t emerge frm the ribsme during prtein synthesis) - Secretry prteins cntain a signal sequence at the N- terminus that directs the emerging plypeptide and ribsme t the ER membrane - Plypeptide mves int cisternal space f ER thrugh a prtein-lined, aqueus channel in ER membrane **ctranslatinally *As the signal sequence emerges frm the ribsme, the hydrphbic signal sequence is recgnized by a signal recgnitin particle (SRP) - SRP: binds signal sequence and ribsme arresting further synthesis f plypeptides - Bund SRP: serves as a tag that enables the entire cmplex t bind specifically t the cystlic surface f ER membrane *Cellular membranes are asymmetric: 2 phphlipid layers if a membrane have different cmpsitins Synthesis f Membrane Lipids - Mst membrane lipids are synthesized entirely within ER Exceptins are: Sphingmyelin and glyclipids (synthesis begins in ER and cmpleted in glgi cmplex; sme unique lipids f mitchndrial and chlrplast membranes (synthesized by enzymes that reside in thse membranes Glgi Cmplex - Divided int several functinally distinct cmpartments arranged alng an axis frm the cis r entry phase clsest t ER - Plays a key rle in the assembly f the carbhydrate cmpnent f glycprteins and glyclipids - Cis-mst phase cmpsed f an intercnnected netwrk f tubules (CGN) CGN (Cis-Glgi netwrk) Srting statin that distinguishes between prteins t be shipped back t ER - Medial glgi Intermediary cisternae - Trans r exit face Fund at ppsite end f the stack f the glgi apparatus TGN (trans-glgi netwrk) Srting statin where prteins are segregated int different types f vesicles heading either t the plasma membrane r the varius intracellular destinatin Mvement f materials thrugh the Glgi cmplex is facilitated by vesicular transprt which is facilitated by cated vesicles 3 best studied cated vesicles - COPII cated vesicles: mve materials frm ER frward t the ERGIC - COPI cated vesicles: mve materials in a retrgrade directin (frm ERGIC and Glgi stack backward twar d the ER; and frm the Glgi cisternae backward t the cis- Glgi cisternae - Clathrin-cated vesicles: mve materials frm the TGN t endsmes, lyssmes, and plant vacule; als mve materials frm plasma membrane t cytplasmic cmpartments alng the endcytic pathway Additinal terms t remember: - VSV: vesicular stmatitis virus (used in GFP) *Viruses are useful because they infected cells int factries fr the prductin f viral prteins, which are carried like any ther prtein carg thrugh the bisynthetic pathway - Hmgenize: break-up f cells - Mutant: an rganism r cultured cells whse chrmsmes cntain ne r mre genes that encde abnrmal prteins; in absence f vesicle frmatin, mutant cells accumulate an expanded ER - Temperature-sensitive mutants: functin nrmally at a reduced ( permissive) temp but nt at an elevated (restrictive) temp - Subcellular fractinatin: vesicles derived frm different rganelles have different prperties - Micrsmes: hetergenus cllectin f similar sized vesicles frmed by membranus vesicles derived frm endmembrane system primarily ER and glgi cmplex) - Cell-free systems: d nt cntain whle cells; prvide a wealth f inf abut bilgical prcesses that were impssible t study within the cmplex envirnment f intact cells - RNA interference (RNAi): a prcess in which cells prduce small RNAs (sirnas) that bind t specific mrnas and inhibit the translatin f these mrnas int prteins - Translcn: prtein-lined channel embedded in ER membrane thrugh which nascent plypeptide is able t mve in its passage frm the ribsme t ER lumen - GTP binding prteins (G prteins): play key regulatry rles in many diff cellular prcesses; like mlecular switches G prtein turns prcess ON while hydrlysis f G prtein turns prcess OFF - Signal peptidase: prtelytic enzyme that remves N- terminal prtin cntaining the signal peptide frm mst nascent plypeptides - Flippases:lipid mlecules that are flipped int ppsite leaflet thrugh enzyme actins - Glycsyltransferases:large family f membrane-bund enzymes that catalyzesadditin f sugars t an ligsaccharide chain/ simply catalyze the glycsylatin prcesses in glgi THE GOLGI COMPLEX - Camill Glgi Italian bilgist Discvered a darkly stained reticular netwrk lcated near the cell nucleus GOLGI COMPLEX - Glgi Cmplex Has a characteristic mrphlgy cnsisting f flattened, disk-like, membranus cisternae with dilated rims and assciated vesicles and tubules - Cisternae Diameter typically 0.5µm t 1.µm Arranged in rderly stack and are curved - Glgi stack Typically cntains fewer than 8 cisternae An individual cell may cntain several distinct stacks, depending n the cell type Mammalian cells Glgi stacks are intercnnected t frm a single, large ribbn-like cmplex situated adjacent t the cell s nucleus - Divided int several functimality distinct cmpartments arranged alng an axis frm cis (entry face clsest t ER) t the trans (exit face at the ppsite end f the stack) - cis-mst face f the rganelle Cmpsed f intercnnected netwrk f tubules (cis Glgi netwrk - CGN) CGN srting statin that distinguishes between prteins t be shipped back t ER and thse allwed t prceed t the next Glgi statin - Cmpsed f a series f large flattened cisternae cis cisternae medial cisternae trans cisternae - trans-mst face f the rganelle cntains a distinct netwrk f tubules and vesicles (trans Glgi netwrk TGN) TGN srting statin where prteins are segregated int different types f vesicles heading either t the plasma membrane r t varius intracellular destinatin - The membranus elements f the Glgi cmplex are thught t be supprted mechanically by a peripheral membrane skeletn r scaffld cmpsed f a variety f prteins, including members f the spectrin, ankyrin, and actin families - The Glgi scaffld may be physically linked with mtr prteins that direct the mvement f vesicles and tubules entering and exiting the Glgi cmplex. - A separate grup f fibrus prteins are thught t frm a Glgi matrix, that plays a key rle in the disassembly and reassembly f the Glgi cmplex during mitsis.

3 Vesicles can mve in a backward (retrgrade) directin, that is, frm a trans dnr membrane t a cis acceptr membrane The cmpsitin f an individual Glgi cisterna can change ver time frm ne that cntains early (cis) Glgi resident prteins t ne that cntains late (trans) Glgi resident prteins. Glycsylatin in the Glgi Cmplex - Glgi cmplex plays a key rle in the assembly f the carbhydrate cmpnent f glycprteins and glyclipids - Mst f the manrse residues are als remved frm the cre ligsaccharides and ther sugars are added sequentially by varius glycsyltransferases - The sequence in which sugars are incrprated int ligsaccharides is determined by the spatial arrangement f the specific glycsyltransferases that cme int cntact with the newly synthesized prtein as it mves thrugh the Glgi stack (Glgi Cmplex RER) - Sialytransferases (enzyme) Places sialic acid at the terminal psitin f the chain in animal cells Lcalized in the trans face f the Glgi stack - Glycsylatin in the ER Assembles a single cre ligsaccharide Prduce carbhydrate dmains remarkable sequence diversity - The Glgi cmplex is als the site f synthesis f mst f a cell s cmplex plysaccharides Glycsaminglycan chains f the prteglycan Pectins and hemicellulse fund in the cell walls f plants Mvement f Material thrugh the Glgi Cmplex - Up until mid-1980s Glgi cisternae were transient structures Glgi cisternae frmed at the cis face f the stack fusin f the membranus carriers frm the ER and ERGIC Each cistern physically mved frm the cis t the trans end f the stack Citernal Maturatin Mdel Each cistern matures int the next cisterna alng the stack - Mid-1980s t mid01990s The cisternae f a Glgi stack remain in place as stable cmpartments Vesicular Transprt Mdel Carg Carg is shuttled thrugh the Glgi stack, frm the CGN t the TGN, in vesicles that bud frm ne membrane cmpartment and fuse with a neighbring cmpartment farther alng the stack Each f the varius Glgi cisternae f a stack has a distinct ppulatin f resident enzymes Large numbers f vesicles can be seen in electrn micrgraphs t bud frm the rims f Glgi cisternae - Cnsensus shifted back t the cisternal maturatin mdel The cisternal maturatin mdel envisins a highly dynamic Glgi cmplex in which the majr elements f the rganelle, the cisternae, are cntinually being frmed at the cis face and dispersed at the trans face Certain materials that are prduced in the endplasmic reticulum and travel thrugh the Glgi cmplex can be shwn t remain within the Glgi cisternae and never appear within Glgiassciated transprt vesicles TYPES OF VESICLE TRANSPORT AND THEIR FUNCTIONS - The bisynthetic pathway f a eukarytic cell cnsists f a series f distinct membrane-bund rganelles that functin in the synthesis, mdificatin, and delivery f sluble and membrane prteins t their apprpriate destinatin in the cell - The dark-staining layer cnsists f a prtein cat frmed frm sluble prteins that assemble n the cytslic surface f the dnr membrane at sites where budding takes place Each cated bud pinches ff t frm a cated vesicle - Functins f prtein cats Act as a mechanical device that causes the membrane t curve and frm a budding vesicle Prvide a mechanism fr selecting the cmpnents t be carried by the vesicle Carg cnsisting f secretry, lyssmal, and membrane prteins t be transprted Machinery required t target and dck the vesicle t the crrect acceptr membrane - Vesicle cat is cmpsed f tw distinct prtein layers: An uter cage r scafflding that frms the framewrk fr the cat an inner layer f adaptrs that binds bth t the uter surface f the lipid bilayer and the membrane s carg - Three best studied cat vesicle COPII-cated vesicles Mve materials frm the ER frward t the ERGIC and Glgi cmplex. COPI-cated vesicles Mve materials in a retrgrade directin Clathrin-cated vesicles Frm the ERGIC and Glgi stack backward tward the ER and Frm trans Glgi cisternae backward t cis Glgi cisternae Mve materials frm the TGN t endsmes, lyssmes, and plant vacules Mve materials frm the plasma membrane t cytplasmic cmpartments alng the endcytic pathway Trafficking frm endsmes and lyssmes. COPII-Cated Vesicles: Transprt Carg frm the ER t the Glgi Cmplex - COPII-cated vesicles mediate the first leg f the jurney thrugh the bisynthetic pathway frm the ER t the ERGIC and Glgi cmplex - COPII cats select and cncentrate certain cmpnents fr transprt in vesicles

4 - Certain integral membrane prteins f the ER are selectively captured because they cntain ER exprt signals as part f their cytslic tail Signals interact specifically with COPII prteins f the vesicle cat - Prteins selected by COPII-cated vesicles include: Enzymes that act at later stages in the bisynthetic pathway, such as the glycsyltransferases f the Glgi cmplex Membrane prteins invlved in the dcking and fusin f the vesicle with the target cmpartment, - Membrane prteins that are able t bind sluble carg - Cells lacking a specific carg receptr typically fail t transprt a specific subset f prteins frm the ER t the Glgi cmplex. - Prteins that nrmally reside in the ER cntain shrt amin acid sequences at their C-terminus that serve as retrieval signals, ensuring their return t the ER if they shuld be accidentally carried frward t the ERGIC r Glgi cmplex. - The retrieval f escaped ER prteins frm these cmpartments is accmplished by specific receptrs that capture the mlecules and return them t the ER in COPIcated vesicles - Sar1 Small G prtein Recruited specifically t the ER membrane Regulatry rle Initiating vesicle frmatin Regulating the assembly f vesicle cat - Resident prteins f the ER cntain amin acid sequences that lead t their retrieval frm the Glgi cmplex if they are accidentally incrprated int a Glgi-bund transprt vesicle. Sluble ER prteins bear the retrieval signal KDEL (lys-asp-glu-leu). Retrieval is accmplished as sluble ER prteins bind t KDEL receptrs residing in the membranus wall f cis Glgi cmpartments. The KDEL receptrs, in turn, bind t prteins f the COPI cat, which allws the entire cmplex t be recycled back t the ER. Beynd the Glgi Cmplex: Srting Prteins at the TGN - The trans Glgi netwrk (TGN) Last stp in the Glgi cmplex Functins as a majr srting statin, directing prteins t varius destinatins - The best understd f the pst-glgi pathways is ne that carries lyssmal enzymes Srting and Transprt f Lyssmal Enzymes - Lyssmal prteins are synthesized n membrane-bund ribsmes f the ER and carried t the Glgi cmplex alng with ther types f prteins - Once in the Glgi cisternae, sluble lyssmal enzymes are specifically recgnized by enzymes that catalyze the twstep additin f a phsphate grup t certain mannse sugars f the N-linked carbhydrate chains COP1 Cated Vesicles: Transprting Escaped Prteins Back t the ER - COPI-cated vesicles accumulate in the presence f a nnhydrlyzable GTP analgue because, similar t their COPII cunterparts, the cat cntains a small membranebending GTP-binding prtein, called Arf1, whse bund GTP must be hydrlyzed befre the cat can disassemble. - COPI-cated vesicles have been mst clearly implicated in the retrgrade transprt f prteins, including the mvement f: Glgi resident enzymes in a trans-t-cis directin ER resident enzymes frm the ERGIC and the Glgi cmplex back t the ER Retaining and Retrieving Resident ER Prteins - Studies suggest that prteins are maintained in an rganelle by a cmbinatin f tw mechanisms: Retentin f resident mlecules that are excluded frm transprt vesicles. Retentin may be based primarily n the physical prperties f the prtein. Retrieval f escaped mlecules back t the cmpartment in which they nrmally reside.

5 - Lyssmal enzymes pssess phsphrylated mannse residues, which act as srting signals - Lyssmal enzymes carrying a mannse 6-phsphate signal are recgnized and captured by mannse 6-phsphate receptrs (MPRs), which are integral membrane prteins that span the TGN membranes - Lyssmal enzymes are transprted frm the TGN in clathrin-cated vesicles - Cats f the vesicles cntain An uter hneycmblike lattice cmpsed f the prtein clathrin, which frms a structural scaffld, An inner shell cmpsed f prtein adaptrs, which cvers the surface f the vesicle membrane that faces the cytsl - Lyssmal enzymes are escrted frm the TGN by a family f adaptr prteins called GGAs - Clathrin-cated vesicles that bud frm the TGN cntain GGA, an adaptr prtein cnsisting f several distinct dmains. One f the GGA dmains binds t the cytslic dmains f membrane prteins, including thse that will ultimately reside in the bundary membrane f the lyssme and als the MPR that transprts lyssmal enzymes. Other GGA dmains bind t Arf1 and t the surrunding cytslic netwrk f clathrin mlecules Srting and Transprt f Nnlyssmal Prteins - Vesicle fusin requires specific interactins between different membranes Vesicles frm the ER Fuse with the ERGIC r cis Glgi netwrk and nt with a trans cistern - Selective fusin is ne f the factrs that ensures a directed flw thrugh the membranus cmpartments f the cell - It is thught that a vesicle cntains specific prteins assciated with its membrane that gvern the mvements and fusin ptential f that vesicle - Steps that ccur between the stages f vesicle budding and vesicle fusin Mvement f the vesicle tward the specific target cmpartment. Membranus vesicles must mve cnsiderable distances thrugh the cytplasm befre reaching their eventual target These types f mvement are mediated largely by micrtubules, which act like railrad tracks carrying carg cntainers alng a defined pathway t a predetermined destinatin Tethering vesicles t the target cmpartment. The initial cntacts between a transprt vesicle and its target membrane, such as a Glgi cisterna, are thught t be mediated by a diverse cllectin f tethering prteins Tw grups f tethering prteins have been described Rd-shaped, fibrus prteins Capable f frming a mlecular bridge between the tw membranes Large multiprtein cmplexes Appear t hld the tw membranes in clser prximity Different tethering prteins initiate fusin between different types f membranes Glgins One class f fibrus tethering prteins Act in and arund the Glgi cmplex May serve as tentacles t reach ut and capture transprt vesicles carrying Glgi-bund carg Rabs Small G prteins Cnfer the specificity between vesicle and target Cycle between an active GTPbund state and an inactive GDP-bund state.gtp-bund Assciate with membranes by a lipid anchr Cnstitute the mst diverse grup f prteins invlved in membrane trafficking. Assciated with different membrane cmpartments Dcking vesicles t the target cmpartment. The membranes f the vesicle and target cmpartment cme int clse cntact with ne anther The key prteins that engage in these interactins are called SNAREs All cntain a segment in their cytslic dmain called a SNARE mtif that cnsists f amin acids capable f frming a cmplex with anther SNARE mtif. SNAREs can be divided functinally int tw categries v-snares which becme incrprated int the membranes f transprt vesicles during budding t-snares which are lcated in the membranes f target cmpartments Fusin between vesicle and target membranes When artificial lipid vesicles (lipsmes) cntaining purified t- SNAREs are mixed with lipsmes cntaining purified v-snares, the tw

6 types f vesicles fuse with ne anther, but nt with themselves Interactins between t- and v-snares are capable f pulling tw lipid bilayers tgether with sufficient frce t cause them t fuse Althugh interactin between v and t- SNAREs is required fr membrane fusin, it is nt sufficient by itself t bring abut fusin within a cell. Excytsis - The fusin f a secretry vesicle r secretry granule with the plasma membrane and subsequent discharge f its cntents - Prbably ccurs n a rather cntinual basis in mst cells - Hwever, the best studied examples f excytsis are thse that ccur during regulated secretin Release f neurtransmitters int the synaptic cleft Membrane fusin prduces an pening thrugh which the cntents f the vesicle r granule are released int the extracellular space - In ther types f cells, excytsis is generally triggered by release f Ca2+ frm cytplasmic stres - Cntact between the vesicle and plasma membranes is thught t lead t the frmatin f a small, prtein-lined fusin pre - When a cytplasmic vesicle fuses with the plasma membrane, the luminal surface f the vesicle membrane becmes part f the uter surface f the plasma membrane, whereas the cytslic surface f the vesicle membrane becmes part f the inner (cytslic) surface f the plasma membrane LYSOSOMES - An animal cell s digestive rganelles - A typical lyssme cntains at least 50 different hydrlytic enzymes prduced in the rugh ER and targeted t these rganelles - Lyssmal enzymes can hydrlyze virtually every type f bilgical macrmlecule - All have their ptimal activity at an acid ph and thus are acid hydrlases. The ph ptimum f these enzymes is suited t the lw ph f the lyssmal cmpartment, which is apprximately Lyssmal membranes als cntain a variety f highly glycsylated integral prteins Carbhydrate chains are thught t frm a prtective lining that shields the membrane frm attack by the enclsed enzymes. - The appearance f lyssme in electrn micrpgrahs is neither distinct nr unifrm - The best studied rle f lyssmes is the breakdwn f materials brught int the cell frm the extracellular envirnment Autphagy - Lyssmes als play a key rle in rganelle turnver The regulated destructin f the cell s wn rganelles and their replacement - Autphagy Prcess An rganelle is surrunded by a duble membrane structure t prduce a dublemembrane sequestering vesicle called an autphagsme - The uter membrane f the autphagsme fuses with a lyssme, generating a structure called an autlyssme, in which bth the inner membrane f the autphagsme and the enclsed cntents are degraded. - The prducts f these degradative reactins are made available t the cell. - It is estimated that 1 t 1.5 percent f the prteins within a healthy liver cell are degraded via autphagy per hur as part f a nrmal prcess f cellular renvatin. - Autphagy prbably evlved in early eukarytic rganisms as a respnse t nutrient deprivatin. - If a ppulatin f cells is placed under starvatin cnditins, a marked increase in autphagy is bserved. - Under these cnditins, cells acquire energy t maintain their life by cannibalizing their wn rganelles - Deletin f certain f these autphagy genes can have serius cnsequences fr embrynic develpment r adult physilgy f mdel rganisms Autphagy helps prtect an rganism against intracellular threats ranging frm abnrmal prtein aggregates t invading bacteria and parasites - Autphagy has als been implicated in the preventin f neurdegeneratin - Autphagy may even play a rle in the preventin f certain types f cancers and slwing the bdy s aging prcess - Once the digestive prcess in the autlyssme has been cmpleted, the rganelle is termed a residual bdy. Depending n the type f cell, the cntents f the residual bdy may be eliminated frm the cell by excytsis, r they may be retained within the cytplasm indefinitely as a lipfuscin granule. THE HUMAN PERSPECTIVE: DISORDERS RESULTING FROM DEFECTS IN LYSOSOMAL FUNCTIONS - Mannse 6-phsphate residues in lyssmal enzymes act as an address fr delivery f these prteins t lyssmes - The discvery f the lyssme address was made in studies f patients with a rare and fatal inherited cnditin knwn as I-cell disease. Many cells in these patients cntain lyssmes that are blated with undegraded materials The secreted enzymes lacked the mannse phsphate residues that are present n the lyssmal enzymes f cells frm nrmal individuals. The I-cell defect was sn traced t the deficiency f an enzyme (N-acetylglucsamine phsphtransferase) required fr mannse phsphrylatin in the Glgi cmplex - In 1965, H. G. Hers f the University f Luvain in Belgium - ffered an explanatin as t hw the absence α-glucsidase culd lead t the develpment f a fatal inherited cnditin knwn as Pmpe disease In the absence f α-glucsidase, undigested glycgen accumulated in lyssmes Causing swelling f the rganelles and irreversible damage t the cells and tissues. - Lyssmal strage disrders Characterized by the deficiency f a single lyssmal enzyme and the crrespnding accumulatin f undegraded substrate

7 The symptms f lyssmal strage diseases can range frm very severe t barely detectable, depending primarily n the degree f enzyme dysfunctin. - Tay- Sachs disease Results frm a deficiency f the enzyme β-nhexsaminidase A, an enzyme that degrades the gangliside GM2 GM2 is a majr cmpnent f the membranes f brain cells, and in the absence f the hydrlytic enzyme, the gangliside accumulates in the blated lyssmes f brain cells, causing dysfunctin. In its severe frm, the disease is characterized by prgressive mental and mtr retardatin, as well as skeletal, cardiac, and respiratry abnrmalities. 1 in 3600 newbrns amng Jews f eastern Eurpean ancestry. - Gaucher s disease A deficiency f the lyssmal enzyme gluccerebrsidase, can be alleviated by enzyme replacement therapy. Infants with Gaucher s disease accumulate large quantities f gluccerebrside lipids in the lyssmes f their macrphages, causing spleen enlargement and anemia - Substrate reductin therapy Small mlecular weight drugs are administered t inhibit the synthesis f the substances that accumulate in the disease 12.7 Plant Cell Vacules - Single membrane-bund, fluid-filled central Vacule - String slutes, macrmlecules (ins, sugars, amin acids, prteins and plysaccharides)& txic cmpunds fr injury caused by herbivre (cyanide cntaining glycsides and glucsinlates) - Islate prducts f metablic reactins - Tnplast membrane that bunds the vacule, cntains number f active transprt systems that pump ins int vacular cmpartment t a cncentratin much higher than that in the cytplasm r extracellular fluid. - High in cncentratin H20 enters vacule (smsis) - Hydrstatic turgr pressure by vacule prvides mechanical supprt, stretches cell wall during cell grwth - Sites f intracellular digestin, plant vacules have acid hydrlases ph f vacule is maintained at lw value by V- type H+ - ATPase w/in the tnplast that pumps prtns int the vacular fluid Prteins f lyssme are synthesised n membrane-bund ribsmes f RER, transprted thrugh the Glgi Cmplex and srted at the trans face f the Glgi Endcytic Pathway: Mving Membrane and Materials int the Cell Interir - Endcytsis cell internalizes cell-surface receptrs and bund extracellular ligands - Phagcytsis describes the uptake f particulate matter - Endcytsis Bulk-phase (pincytsis) nn-specific uptake f extracellular fluids, large r small. Remves prtins f Plasma Membrane bet. cell surface and interir cmpartments Receptr mediated brings abut the uptake f specific extracellular macrmlecules (ligands) fllwing their binding t receptrs n the external surface f the plasma membrane Receptr-Mediated Endcytsis and the Rle f Cated Pits (PM Plasma Membrane) - Means fr selective and efficient uptake f macrmlecules - Have receptrs fr diff. types f ligands (hrmnes, grwth factrs, enzymes and bld brne prteins carrying nutrients) - Substances that enter a cell by means f clathrin-mediated RME becme bund t receptrs that cllect in specialized dmains f the PM, knwn as cated pits. Cated pits sites where surface is indented and PM is cvered n its cytplasmic face by clathrin. Invaginate int cytplasm and pinch free f the PM t frm cated vesicles Cated pit derived frm structure f clathrin building blcks - Each clathrin cat ml. cnsists f three heavy chains and three light chains, jined tgether at the center t frm a three-legged assembly called triskellin - Cated vesicles that frm during endcytsiscntain layer f adaptrs between clathrin lattice and surface f the vesicle facing the cytsl - AP2 best studied adaptr, incrprated int the vesicles that bud frm the PM cntain multiple subunits having diff. functins - The u subunit f AP2 adaptrs engage the cytplasmic tails f PM receptrs leading t the cncentratin f these selected receptrs and their bund carg mlecules int emerging cated vesicle - In cntrast, B-adaptin subunit f AP2 adaptrs binds and recruits the clathrin mlecules f the velying lattice. - CpII and clathrin cated vesicles have uter gemetric scaffld and inner layer f adaptr prteins - Outer gemetric scafflds subunits f clathrin lattice verlap, COPII lattice d nt verlap - Vertex f COPII cate frmed by fur edges rather than 3 in clathrin cat - Dynamin large GTP-binding prtein required fr release f clathrin-cated vesicle frm membrane n w/c it frms. - Self-assembles int helical cllar arund neck f invaginated cated pit. - Hydrlysis f the bund GTP by plymerized dynamin ml induces twisting mtin in the dynamin helix that severs the cated vesicle frm the plasma membrane. - Acts as an enzyme capable f utilizing the chemical energy f GTP t generate mechanical fces. - ATPase Hsc70 helps in dissc. f clathrin cat frm vesicle recruited by clathrin cat by a cfactr, auxilin The Rle f Phsphinsitides in the Frmatin f Cated Vesicles - Phsphate grups can be added t diff. psitins f the sugar ring f the Phsphatidylinsitl (PI), cnverting them int phsphinsitl (PI) - Phsphrylated rings f these phsphinsitides reside at the surface f the membrane t be recgnized and bund by particular prteins - Diff. phsphinsitides cncentrated in diff. membrane cmpartments = unique surface identity - PI(4,5)P2 has dynamic regulatry rle bec. can be rapidly frmed and destryed enzymes that are lcalized at particular places & times within the cell Endcytic Pathway - Rute f mlecules taken int a cell by endcytsi - Receptrs subjected t endcytsis: Husekeeping receptrs resp. fr uptake f materials used by the cell Ex. Transferrin, Lw density lipprtein mediate delivery t cells f irn and chlesterl respectively Signaling receptrs binding extracellular ligands that carry messages that change the activities f the cell. - Ligands include insulin, grwth factrs (EGF) that bind t surface receptr and signal a physilgic respnse inside the cell - Endcytsis f 1 st grup f receptrs leads t delivery f the bund materials (irn and chlesterl), t the cell and return f the receptr t the cell surface fr uptake - Endcytsis f 2 nd grup f receptrs ften leads t the destructin f the receptr, a prcess called receptr dwnregulatin, which has the effect f reducing the sensititivity f the cell t further stimulatin by the hrmne r grwth factr. - Receptr dwn-regulatin is a mechanism by w/c cells regulate their ability t respnd t extracellular messengers.

8 - Signaling receptrs endcytsis and destructin by cvalent attachment f a tag t cytplasmic tail f the receptr while residing at cell surface - Tag small prtein called ubiquitin. - Fllwing internalizatin, vesicle-bund materials transprted t endsmes w/c represent distributin centers. - Fluid in the lumen is acidified by H+ - ATPase in the bundary membrane - Endsmes divided int tw classes: Early near the peripheral f cell Late clser t nucleus multivesicular bdies (MVBs) - Receptrs taken up by endcytsis transprted in vesicles t an early endsme serves as srting statin that directs diff. types f receptrs and ligands alng diff. pathways. - Husekeeping recepters dissc. frm bund ligands as result f high H+ cncentratin f early endsmes. - Receptrs cncentrated int specialized tubular cmpartments f early endsme, represent recycling centers. Vesicles that bud frm these tubules carry receptrs back t the PM fr addtl runds f endcytsis - In cntrast, released ligands becme cncentrated int a srting cmpartment befre being dispatched t a late endsme and befre dispatched t a late endsme and t a lyssme where final prcessing ccurs - Signaling receptrs w/ ubiquitin tags d nt recycle back t membrane. - Ubiquitinated receptrs are sequestered int pp. f small, spherical vesicles that crwd the interir f late endsme. - Vesiculatin prcess (4 diff. ESCRT cmplexes act in seq. 1. srt ubiquitinated receptrs int a cluster w/in late endsmal membrane 2. cause patch f membrane t invaginate as bud int lumen f late endsme 3. sever neck f the invaginatin t release newly frmed intraluminal vesicle LDLs and Chlesterl Metablism - Prvides animal cells w/ exgenus chlesterl (part f PM, precursr f sterid). - Chlesterl hydrphbic ml. transprted in bld as part f hugh lipprtein cmplexes lw-density lipprtein) - Each LDL cntains central cre f 1500 chlesterl ml esterified t lng-chain fatty acids. - Cre surrunded by single layer f phsphlipids w/ 1 cpy f large prtein called aplipprtein B-10 (binds t LDL) - LDL transprted t PM, cncentrated in cated pits, even absence f LDL ligand - Receptrs ready t take up bld-brne lipprteins - Once LDL particles bund t cated pit, pit invaginates t frm cated vesicle, claithrin cat disassembled and LDL recpetrs pass thrugh early endsmes and back t PM. - Meanwhile, LDL particles are delivered t late endsmes and lyssmes, where prtein cmpnent is degraded and chlesterl is deesterified and used by cell in membrane assembly r ther metablic prcesses. - Niemann-pick type C diesease lack ne prteins req. t transfer t chlesterl ut f lyssmes. - Resulting accumulatin f chlesterl lead t nerve degeneratin and death during early childhd. - LDL level in bld rel. t athersclersis frmatin f plaques in inner lining f arteries that reduce flw f bld thrugh the vessel and act as sites fr frmatin f bld clts - Bld clts that blck crnary arteries lead t myccardial infarctin (heart attack) - Lwering LDL levels thrugh statins the blck HMG CA reductase (synthesis f chlesterl) Lwer bld chlesterl risk f heart attack is reduced - HDLs (high-density lipprteins) cntain diff. prtein (aplipprtein A-I) play diff. physilgic rle in bdy - Carries chlesterl in ppsite directin - Excess chlesterl transprte dut f PM f bdy cells circulating HDL particles, w/c carry chlesterl t liver fr extractin - LDL serves t carry chlesterl ml frm liver, synthesized packaged thrugh the bld t the bdy s cells - High levels f HDL assc. with decreased risk (gd chlesterl) - Small mlecular weight CETP inhibitrs increase HDL levels - PCSK9 prtelytic enzyme that destrys LDL receptrs in liver - Reduced LDL chlesterl levels decreased heart disease Phagcytsis - Uptake f large particles frm envirnment - Single cell prtists (amebas and ciliates) - Trap fd particles and smaller rganisms, enclsing in flds in PM - Fuse t prduce vacule (phagsme) pinchess inwardly frm PM - Phagsme fuses w/ lyssme, digested w/in resulting phaglysme - In animals, prtective mechanism prfessinal phagcytes Macrphages and neutrphils - Wander thrugh bld and tissues phagcytizing invading rganisms, damaged and dead cells and debris - Recgnized and bund by recepters n surface f phagcyte prir t uptake - Once inside phagcyte, micrrganisms killed by lyssmal enzymes r xygen free radicals generated w/in lumen f phagsme - Driven by cntractile activities f actin-cntaining micrfilaments that underlie the plasma membrane - Nt all bacteria ingested by phagcytic cells are destryed. - Sme hijack phagcytic machinery t survive - Mycbacterium tuberculsis, (tuberculsis) taken int cytplasm f macrphage by phagcytsis, bacterium is able t inhibit fusin f phagsme w/ lyssme - Phagsme becmes highly aciding, bacterium appears able t maintain ph despite lwerd ph f surrunding medium. - Cxiella burnetii enclsed in phagsme fuse w/ lyssme but acidic envirnment nr lyssmal enzymes cannt destry it - Listeria mncytgenes, causes meningits, prduces prteins that destry integrity f the lyssmal membrane, allwing bacterium t escape int cell s cytsl. POSTTRANSLATIONAL UPTAKE OF PROTEINS BY PEROXISOMES, MITOCHONDRIA AND CHLOROPLAST - The divisin f the cntents f a cell int large numbers f cmpartments presents many rganizatinal challenges t the cell s prtein-trafficking machinery - Prtein trafficking within a eukarytic cell Srting signals, such as the signal peptide f secreted prteins r mannse-phsphate grups f lyssmal enzymes Receptrs that recgnize these signals and deliver prteins cntaining them t the prper cmpartment - Fur f the cell s majr rganelles nucleus, mitchndria, chlrplasts, and perxismes imprt prteins thrugh ne r mre uter bundary membranes Uptake f Prteins int Perxismes - Perxismes are very simple rganelles having nly tw subcmpartments The bundary membrane The internal matrix - Prteins destined fr a perxisme pssess a perxismal targeting signal, either a PTS fr a perxismal matrix prtein r an mpts fr a perxismal membrane prtein - Perxismes are smehw able t imprt perxismal matrix prteins in their native, flded cnfrmatin, even thse that cnsist f several subunits Uptake f Prteins int Mitchndria - Mitchndria have fur subcmpartments int which prteins can be delivered: An uter mitchndrial membrane (OMM) Inner mitchndrial membrane (IMM) Intermembrane space Matrix - Rughly 99 percent f the rganelle s prteins are encded by the nuclear genme, synthesized in the cytsl, and imprted psttranslatinally - Mitchndrial prteins cntain signal sequences that target them t their hme base. - The plypeptide is targeted t a mitchndrin by a targeting sequence, which is lcated at the N-terminus in the matrix prtein and is lcated internally in mst inner membrane prteins. Cytslic Hsp70 mlecules unfld the

9 plypeptides prir t their entry int the mitchndrin. The prteins are recgnized by membrane receptrs (red transmembrane prteins) and translcated thrugh the OMM by way f pres in the TOM cmplex f the OMM. Mst integral prteins f the IMM are directed t the TIM22 cmplex f the IMM, which steers them int the lipid bilayer f the IMM. Mitchndrial matrix prteins are translcated thrugh the TIM23 cmplex f the IMM. Once the prtein enters the matrix, it is bund by a mitchndrial chaperne, which may either pull the plypeptide int the matrix r act like a Brwnian ratchet t ensure that it diffuses int the matrix. Once in the matrix, the unflded prtein assumes its native cnfrmatin with the help f Hsp60 chaperne. The presequence is remved enzymatically. Uptake f Prteins int Chlrplast - Chlrplasts have six subcmpartments int which prteins can be delivered: Inner envelpe membrane Outer envelpe membrane Intervening intermembrane space Strma Thylakid membrane Thylakid lumen - Chlrplast and mitchndrial imprt mechanisms exhibit many similarities, althugh their translcatin machineries have evlved independently Tthe vast majrity f chlrplast prteins (apprximately 3000 in higher plants) are imprted frm the cytsl, The uter and inner envelpe membranes cntain distinct translcatin cmplexes (Tc and Tic cmplexes, respectively) that wrk tgether during imprt Chapernes aid in the unflding f the plypeptides in the cytsl and flding f the prteins in the chlrplast Mst prteins destined fr the chlrplast are synthesized with a remvable N-terminal sequence (termed the transit peptide) that is highly variable in length and sequence. - Prteins encded by nuclear genes are synthesized in the cytsl and imprted thrugh prtein-lined pres in bth membranes f the uter chlrplast envelpe. Prteins destined fr the strma cntain a strma targeting dmain at their N-terminus, whereas prteins destined fr the thylakid cntain bth a strma-targeting dmain and a thylakid-transfer dmain at their N-terminus. Strmal prteins remain in the strma fllwing translcatin thrugh the uter envelpe and remval f their single targeting sequence. The presence f the thylakid transfer dmain causes thylakid prteins t be translcated either int r cmpletely thrugh the thylakid membrane. A number f the prteins f the thylakid membrane are encded by chlrplast genes and synthesized by chlrplast ribsmes that are bund t the uter surface f the thylakid membrane. - 1b