please refer to the file
BIOC4610 Problem-solving Questions for Continuous Assessment (7.5%) Dr. Jeff Shih-Chieh Ti 1. The Golgi apparatus sorts newly synthesized acid hydrolases into vesicles for delivery to endosomes and lysosomes, away from the exocytic pathway. The sorting is affected by XPR, a type I integral membrane protein with (1) a luminal domain for capturing cargo enzymes, and (2) a cytoplasmic tail for sorting to endosomes and lysosomes. XPR localization to Golgi sub-fractions was assessed with use of three different cell lines- rat hepatocytes (clone 9), rat kidney epithelial cells (NRK) and Chinese hamster ovary (CHO-K1). Results [Brown & Farquhar (1987) PNAS 84: 9001-9005] are shown in Figure 1 and 2 below. Figure 1. Rat clone 9, NRK and CHO-K1 cells were separately subjected to sucrose density gradient ultra-centrifugation. Profiles of XPR-immunoreactivity (—), enzyme activity of ?-mannosidase (- - - ; a medial Golgi marker) and that of galactosyltransferase (…..; a trans Golgi marker) are shown for each of the cell lines. Figure 2. Golgi subfractions were solubilized in a mix of detergents and then subjected to immunoprecipitation with use of anti-XPR. The immunoprecipitates were subjected to SDS-PAGE. Electrophoreto-grams were visualized by fuorograophy. Fraction numbers in the fluorograms correspond to those in Figure 1. a) What moiety in acid hydrolases is capture by the reaction domain of XPR? (1 mark) Write a key enzyme reaction responsible for transfer of this moiety to acid hydrolases. (3 mark) Does the reaction occur in the cis, medial or trans Golgi cisternae? (1 mark) b) By reference to Figures 1 and 2, how do the three cell lines differ in the steady-state distribution of XPR across the stacks of Golgi cisternae? (6 marks) c) Cultures of clone 9 hepatocytes were treated for 3 hr with tunicamycin and then processed for immunoelectron microscopy. XPR-immunoreactivity was dateable in coated vesicles lining up along the cis Golgi cisternae but was not detectable in endosomes and lysosomes. (i) What enzymatic reaction is inhibited by tunicamycin? (2 marks) Predict the consequence of this inhibitory activity on N-glycosylation of nascent polypeptides and subsequent modification reactions that occur in the Golgi. Explain. (3 marks) (ii) What can be inferred regarding signal(s) that trigger forward delivery of XPR to endosomes and lysosomes versus recycling of XPR to the Golgi? (3 marks) 2. Compare co-translational translocation of protein through the ER with post-translational translocation in terms of (i) accessory proteins and (ii) exogenous nucleotide. (6 marks) 9004 Cell Biology: Brown and Farquhar 18 16 14 12 3; _0 Cl: - 0 10 8 6 4 2 0 8 NRK A d 2 it~~~, / \ A if*|*|I I~~~~~A._%" 0 12 10 8 6 4 2 0 Bottom Top Clone 9 I..-I..II< -="" cho="" i'="" ,.....,="" hs/="" 3="" 5="" 7="" 9="" 1l="" 13="" 15="" 17="" 19="" 21="" 23="" fraction="" number="" fig.="" 3.="" subfractionation="" of="" golgi="" from="" clone="" 9,="" nrk,="" and="" cho="" cells.="" total="" microsomal="" membranes="" from="" each="" cell="" type="" were="" separated="" by="" flotation="" in="" 20-50%="" sucrose="" gradients.="" the="" distribution="" of="" man-6-p="" receptors="" (-)="" is="" shown="" relative="" to="" a-mannosidase="" ii="" (---),="" a="" middle="" golgi="" marker="" enzyme,="" and="" galactosyltransferase="" .(.="" ),="" a="" trans="" golgi="" marker.="" the="" top="" and="" bottom="" of="" the="" gradients="" are="" indicated.="" (top)="" in="" golgi="" subfractions="" from="" clone="" 9="" cells,="" man-6-p="" receptors="" are="" concentrated="" in="" a="" single="" peak="" toward="" the="" bottom="" of="" the="" gradient="" (p="1.17" g/ml),="" corresponding="" to="" cis="" golgi="" membranes.="" a-mannosidase="" ii="" and="" galactosyltransferase="" activities="" are="" found="" as="" single="" peaks="" in="" lighter="" fractions="" (p="1.15" and="" 1.13-1.14="" g/ml,="" respectively).="" (middle)="" in="" nrk="" cells,="" significant="" amounts="" ofman-6-p="" receptors="" are="" found="" throughout="" the="" golgi="" subfractions="" with="" a="" major="" peak="" coinciding="" with="" the="" peak="" of="" galactosyltransferase="" (p="1.12" g/ml)="" and="" two="" minor="" peaks="" coinciding="" with="" two="" of="" the="" peaks="" of="" a-mannosidase="" ii="" activity,="" which="" has="" a="" broad="" distribution="" in="" these="" cells.="" (bottom)="" in="" the="" case="" of="" cho="" cells,="" man-6-p="" receptors="" are="" concentrated="" in="" lighter="" fractions="" corresponding="" to="" trans="" golgi="" mem-="" branes="" because="" they="" occur="" in="" a="" single="" major="" peak="" (p="1.12" g/ml)="" that="" overlaps="" but="" is="" of="" slightly="" lower="" density="" than="" the="" peak="" of="" galactosyl-="" transferase="" activity.="" a-mannosidase="" ii="" is="" found="" as="" a="" single="" major="" peak="" of="" higher="" density="" (p="1.13-1.14" g/ml).="" located="" in="" the="" fractions="" containing="" the="" peak="" of="" a-mannosidase="" ii="" activity="" (p="1.13-1.14" g/ml).="" thus,="" in="" cho="" cells="" man-6-p="" receptors="" are="" concentrated="" in="" light="" golgi="" subfractions="" corre-="" sponding="" to="" trans="" golgi="" elements="" and="" in="" membranes="" of="" even="" lower="" density.="" these="" results="" are="" in="" accord="" with="" their="" immuno-="" cytochemical="" localization="" to="" trans="" golgi="" cisternae="" (fig.="" 2c="" and="" ref.="" 11).="" discussion="" considerable="" attention="" has="" been="" devoted="" to="" attempting="" to="" identify="" the="" most="" proximal="" location="" along="" the="" exocytic="" or="" so-called="" endoplasmic="" reticulum-to-plasmalemma="" pathway="" c="" lone="" 9="" nrk="" cho="" 1="" 3="" 5="" 7="" 9="" 11="" 13="" 15="" 17="" 19="" 21="" 23="" fig.="" 4.="" fluorogram="" of="" man-6-p="" receptors="" immunoprecipitated="" from="" the="" gradients="" in="" fig.="" 3,="" showing="" the="" differences="" in="" the="" distri-="" bution="" of="" receptors="" among="" golgi="" subfractions="" in="" different="" cell="" types.="" the="" fractions="" correspond="" to="" those="" in="" fig.="" 3.="" in="" golgi="" subfractions="" obtained="" from="" clone="" 9="" cells,="" man-6-p="" receptors="" are="" concentrated="" in="" dense="" (cis)="" subfractions="" (lanes="" 9-11).="" in="" nrk="" cells="" they="" have="" a="" broader="" distribution="" (lanes="" 13-21),="" and="" in="" cho="" cells="" they="" are="" more="" concentrated="" in="" lighter="" (trans)="" golgi="" subfractions="" (lanes="" 18-24).="" where="" lysosomal="" enzymes="" are="" sorted="" for="" targeting="" to="" lyso-="" somes.="" virtually="" all="" studies="" in="" which="" man-6-p="" receptors="" have="" been="" localized="" are="" in="" agreement="" that="" the="" golgi="" complex="" contains="" a="" high="" concentration="" of="" 215-kda="" man-6-p="" receptors="" and="" is="" the="" site="" where="" lysosomal="" enzymes="" are="" sorted="" and="" exit="" the="" secretory="" pathway="" (9-18).="" there="" has="" been="" no="" agreement,="" however,="" on="" the="" distribution="" of="" 215-kda="" man-6-p="" receptors="" within="" the="" golgi="" complex.="" by="" immunocytochemistry="" they="" have="" been="" variously="" reported="" to="" be="" concentrated="" in="" cis="" cisternae="" (9,="" 17),="" in="" trans="" cisternae="" (11)="" including="" so-called="" gerl="" or="" trans="" golgi="" reticular="" elements,="" or="" in="" all="" cisternae="" of="" the="" golgi="" stack="" (12,="" 13,="" 18).="" since="" the="" reports="" were="" from="" investigators="" using="" different="" techniques="" and="" cell="" types,="" it="" was="" impossible="" to="" know="" whether="" the="" discrepancies="" were="" due="" to="" differences="" in="" the="" immunocytochemical="" procedures="" used="" (i.e.,="" immunoperoxidase="" techniques="" vs.="" immunogold="" local-="" ization="" on="" ultrathin="" frozen="" sections)="" or="" whether="" they="" were="" due="" to="" true="" differences="" in="" the="" distribution="" of="" man-6-p="" receptors="" from="" one="" cell="" type="" to="" another.="" to="" resolve="" this="" problem,="" we="" have="" determined="" the="" distri-="" bution="" of="" 215-kda="" man-6-p="" receptors="" within="" golgi="" subfrac-="" tions="" prepared="" from="" three="" cell="" types="" and="" have="" compared="" the="" results="" to="" those="" obtained="" by="" immunoperoxidase="" localization="" of="" the="" receptors="" in="" situ.="" two="" main="" results="" emerged.="" first,="" the="" distribution="" of="" man-6-p="" receptors="" within="" golgi="" subfractions="" prepared="" from="" the="" three="" cell="" types="" was="" found="" to="" be="" quite="" different.="" receptors="" were="" concentrated="" in="" heavy="" (cis)="" golgi="" subfractions="" in="" clone="" 9="" cells,="" were="" concentrated="" in="" light="" (trans)="" subfractions="" in="" cho="" cells,="" and="" were="" broadly="" distrib-="" uted="" among="" subfractions="" of="" heavy,="" light,="" and="" intermediate="" density="" in="" nrk="" cells.="" second,="" the="" findings="" were="" in="" full="" agreement="" with="" those="" obtained="" by="" immunoperoxidase="" local-="" ization="" of="" the="" receptors="" in="" situ="" (figs.="" 2="" a-c),="" where="" the="" receptors="" were="" localized="" in="" cis="" cisternae="" in="" clone="" 9="" cells,="" in="" trans="" cisternae="" in="" cho="" cells,="" and="" broadly="" distributed="" across="" the="" stack="" in="" nrk="" cells.="" thus,="" the="" differences="" in="" receptor="" distribution="" reported="" in="" the="" literature="" appear="" to="" reflect="" true="" differences="" related="" to="" cell="" type="" rather="" than="" limitations="" of="" certain="" immunocytochemical="" procedures.="" in="" this="" as="" well="" as="" in="" our="" earlier="" studies="" (17,="" 18),="" variations="" in="" distribution="" were="" demonstrated="" by="" using="" the="" same="" immunocytochemical="" pro-="" cedure.="" differences="" in="" man-6-p="" receptor="" distribution="" in="" golgi="" subfractions="" prepared="" from="" different="" cell="" types="" also="" have="" been="" reported;="" they="" were="" found="" to="" be="" concentrated="" in="" middle="" and="" light="" membrane="" subfractions="" in="" bw="" 5417="" lymphocytes="" (15)="" but="" were="" present="" in="" both="" cis="" and="" trans="" golgi="" subfractions="" and="" absent="" from="" middle="" golgi="" subfractions="" prepared="" from="" rat="" liver="" (16).="" how="" can="" the="" differences="" in="" receptor="" distribution,="" especially="" the="" cis="" localization,="" be="" explained?="" the="" simplest="" explanation="" is="" that="" there="" may="" be="" differences="" in="" man-6-p-mediated="" lyso-="" proc.="" natl.="" acad.="" sci.="" usa="" 84="" (1987)="" 9004="" cell="" biology:="" brown="" and="" farquhar="" 18="" 16="" 14="" 12="" 3;="" _0="" cl:="" -="" 0="" 10="" 8="" 6="" 4="" 2="" 0="" 8="" nrk="" a="" d="" 2="" it~~~,="" \="" a="" if*|*|i="" i~~~~~a._%"="" 0="" 12="" 10="" 8="" 6="" 4="" 2="" 0="" bottom="" top="" clone="" 9="">< - cho i' ,....., hs/ 3 5 7 9 1l 13 15 17 19 21 23 fraction number fig. 3. subfractionation of golgi from clone 9, nrk, and cho cells. total microsomal membranes from each cell type were separated by flotation in 20-50% sucrose gradients. the distribution of man-6-p receptors (-) is shown relative to a-mannosidase ii (---), a middle golgi marker enzyme, and galactosyltransferase .(. ), a trans golgi marker. the top and bottom of the gradients are indicated. (top) in golgi subfractions from clone 9 cells, man-6-p receptors are concentrated in a single peak toward the bottom of the gradient (p = 1.17 g/ml), corresponding to cis golgi membranes. a-mannosidase ii and galactosyltransferase activities are found as single peaks in lighter fractions (p = 1.15 and 1.13-1.14 g/ml, respectively). (middle) in nrk cells, significant amounts ofman-6-p receptors are found throughout the golgi subfractions with a major peak coinciding with the peak of galactosyltransferase (p = 1.12 g/ml) and two minor peaks coinciding with two of the peaks of a-mannosidase ii activity, which has a broad distribution in these cells. (bottom) in the case of cho cells, man-6-p receptors are concentrated in lighter fractions corresponding to trans golgi mem- branes because they occur in a single major peak (p = 1.12 g/ml) that overlaps but is of slightly lower density than the peak of galactosyl- transferase activity. a-mannosidase ii is found as a single major peak of higher density (p = 1.13-1.14 g/ml). located in the fractions containing the peak of a-mannosidase ii activity (p = 1.13-1.14 g/ml). thus, in cho cells man-6-p receptors are concentrated in light golgi subfractions corre- sponding to trans golgi elements and in membranes of even lower density. these results are in accord with their immuno- cytochemical localization to trans golgi cisternae (fig. 2c and ref. 11). discussion considerable attention has been devoted to attempting to identify the most proximal location along the exocytic or so-called endoplasmic reticulum-to-plasmalemma pathway c lone 9 nrk cho 1 3 5 7 9 11 13 15 17 19 21 23 fig. 4. fluorogram of man-6-p receptors immunoprecipitated from the gradients in fig. 3, showing the differences in the distri- bution of receptors among golgi subfractions in different cell types. the fractions correspond to those in fig. 3. in golgi subfractions obtained from clone 9 cells, man-6-p receptors are concentrated in dense (cis) subfractions (lanes 9-11). in nrk cells they have a broader distribution (lanes 13-21), and in cho cells they are more concentrated in lighter (trans) golgi subfractions (lanes 18-24). where lysosomal enzymes are sorted for targeting -="" cho="" i'="" ,.....,="" hs/="" 3="" 5="" 7="" 9="" 1l="" 13="" 15="" 17="" 19="" 21="" 23="" fraction="" number="" fig.="" 3.="" subfractionation="" of="" golgi="" from="" clone="" 9,="" nrk,="" and="" cho="" cells.="" total="" microsomal="" membranes="" from="" each="" cell="" type="" were="" separated="" by="" flotation="" in="" 20-50%="" sucrose="" gradients.="" the="" distribution="" of="" man-6-p="" receptors="" (-)="" is="" shown="" relative="" to="" a-mannosidase="" ii="" (---),="" a="" middle="" golgi="" marker="" enzyme,="" and="" galactosyltransferase="" .(.="" ),="" a="" trans="" golgi="" marker.="" the="" top="" and="" bottom="" of="" the="" gradients="" are="" indicated.="" (top)="" in="" golgi="" subfractions="" from="" clone="" 9="" cells,="" man-6-p="" receptors="" are="" concentrated="" in="" a="" single="" peak="" toward="" the="" bottom="" of="" the="" gradient="" (p="1.17" g/ml),="" corresponding="" to="" cis="" golgi="" membranes.="" a-mannosidase="" ii="" and="" galactosyltransferase="" activities="" are="" found="" as="" single="" peaks="" in="" lighter="" fractions="" (p="1.15" and="" 1.13-1.14="" g/ml,="" respectively).="" (middle)="" in="" nrk="" cells,="" significant="" amounts="" ofman-6-p="" receptors="" are="" found="" throughout="" the="" golgi="" subfractions="" with="" a="" major="" peak="" coinciding="" with="" the="" peak="" of="" galactosyltransferase="" (p="1.12" g/ml)="" and="" two="" minor="" peaks="" coinciding="" with="" two="" of="" the="" peaks="" of="" a-mannosidase="" ii="" activity,="" which="" has="" a="" broad="" distribution="" in="" these="" cells.="" (bottom)="" in="" the="" case="" of="" cho="" cells,="" man-6-p="" receptors="" are="" concentrated="" in="" lighter="" fractions="" corresponding="" to="" trans="" golgi="" mem-="" branes="" because="" they="" occur="" in="" a="" single="" major="" peak="" (p="1.12" g/ml)="" that="" overlaps="" but="" is="" of="" slightly="" lower="" density="" than="" the="" peak="" of="" galactosyl-="" transferase="" activity.="" a-mannosidase="" ii="" is="" found="" as="" a="" single="" major="" peak="" of="" higher="" density="" (p="1.13-1.14" g/ml).="" located="" in="" the="" fractions="" containing="" the="" peak="" of="" a-mannosidase="" ii="" activity="" (p="1.13-1.14" g/ml).="" thus,="" in="" cho="" cells="" man-6-p="" receptors="" are="" concentrated="" in="" light="" golgi="" subfractions="" corre-="" sponding="" to="" trans="" golgi="" elements="" and="" in="" membranes="" of="" even="" lower="" density.="" these="" results="" are="" in="" accord="" with="" their="" immuno-="" cytochemical="" localization="" to="" trans="" golgi="" cisternae="" (fig.="" 2c="" and="" ref.="" 11).="" discussion="" considerable="" attention="" has="" been="" devoted="" to="" attempting="" to="" identify="" the="" most="" proximal="" location="" along="" the="" exocytic="" or="" so-called="" endoplasmic="" reticulum-to-plasmalemma="" pathway="" c="" lone="" 9="" nrk="" cho="" 1="" 3="" 5="" 7="" 9="" 11="" 13="" 15="" 17="" 19="" 21="" 23="" fig.="" 4.="" fluorogram="" of="" man-6-p="" receptors="" immunoprecipitated="" from="" the="" gradients="" in="" fig.="" 3,="" showing="" the="" differences="" in="" the="" distri-="" bution="" of="" receptors="" among="" golgi="" subfractions="" in="" different="" cell="" types.="" the="" fractions="" correspond="" to="" those="" in="" fig.="" 3.="" in="" golgi="" subfractions="" obtained="" from="" clone="" 9="" cells,="" man-6-p="" receptors="" are="" concentrated="" in="" dense="" (cis)="" subfractions="" (lanes="" 9-11).="" in="" nrk="" cells="" they="" have="" a="" broader="" distribution="" (lanes="" 13-21),="" and="" in="" cho="" cells="" they="" are="" more="" concentrated="" in="" lighter="" (trans)="" golgi="" subfractions="" (lanes="" 18-24).="" where="" lysosomal="" enzymes="" are="" sorted="" for="">