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 mem
ane 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 XXXXXXXXXXPNAS 84: XXXXXXXXXX] 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 co
espond 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)
) 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 infe
ed 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 Farquha
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_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
XXXXXXXXXX1l XXXXXXXXXX
Fraction Numbe
FIG. 3. Subfractionation of Golgi from clone 9, NRK, and CHO
cells. Total microsomal mem
anes 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
eceptors are concentrated in a single peak toward the bottom of the
gradient (p = 1.17 g/ml), co
esponding to cis Golgi mem
anes.
a-Mannosidase II and galactosyltransferase activities are found as
single peaks in lighter fractions (p = 1.15 and XXXXXXXXXXg/ml,
espectively). (Middle) In NRK cells, significant amounts ofMan-6-P
eceptors are found throughout the Golgi subfractions with a majo
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
oad distribution in these
cells. (Bottom) In the case of CHO cells, Man-6-P receptors are
concentrated in lighter fractions co
esponding to trans Golgi mem-
anes 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 = XXXXXXXXXXg/ml).
located in the fractions containing the peak of a-mannosidase
II activity (p = XXXXXXXXXXg/ml). Thus, in CHO cells Man-6-P
eceptors are concentrated in light Golgi subfractions co
e-
sponding to trans Golgi elements and in mem
anes 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 o
so-called endoplasmic reticulum-to-plasmalemma pathway
C lone 9
NRK
CHO
XXXXXXXXXX XXXXXXXXXX
FIG. 4. Fluorogram of Man-6-P receptors immunoprecipitated
from the gradients in Fig. 3, showing the differences in the distri-
ution of receptors among Golgi subfractions in different cell types.
The fractions co
espond to those in Fig. 3. In Golgi subfractions
obtained from clone 9 cells, Man-6-P receptors are concentrated in
dense (cis) subfractions (lanes XXXXXXXXXXIn NRK cells they have a
oader 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
een 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 XXXXXXXXXXThere 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-
ution of 215-kDa Man-6-P receptors within Golgi subfrac-
tions prepared from three cell types and have compared the
esults 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
oadly 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
eceptors were localized in cis cisternae in clone 9 cells, in
trans cisternae in CHO cells, and
oadly distributed across
the stack in NRK cells. Thus, the differences in recepto
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
eported; they were found to be concentrated in middle and
light mem
ane subfractions in BW 5417 lymphocytes (15)
ut were present in both cis and trans Golgi subfractions and
absent from middle Golgi subfractions prepared from rat live
(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 XXXXXXXXXX)
9004 Cell Biology: Brown and Farquha
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
XXXXXXXXXX1l XXXXXXXXXX
Fraction Numbe
FIG. 3. Subfractionation of Golgi from clone 9, NRK, and CHO
cells. Total microsomal mem
anes 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
eceptors are concentrated in a single peak toward the bottom of the
gradient (p = 1.17 g/ml), co
esponding to cis Golgi mem
anes.
a-Mannosidase II and galactosyltransferase activities are found as
single peaks in lighter fractions (p = 1.15 and XXXXXXXXXXg/ml,
espectively). (Middle) In NRK cells, significant amounts ofMan-6-P
eceptors are found throughout the Golgi subfractions with a majo
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
oad distribution in these
cells. (Bottom) In the case of CHO cells, Man-6-P receptors are
concentrated in lighter fractions co
esponding to trans Golgi mem-
anes 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 = XXXXXXXXXXg/ml).
located in the fractions containing the peak of a-mannosidase
II activity (p = XXXXXXXXXXg/ml). Thus, in CHO cells Man-6-P
eceptors are concentrated in light Golgi subfractions co
e-
sponding to trans Golgi elements and in mem
anes 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 o
so-called endoplasmic reticulum-to-plasmalemma pathway
C lone 9
NRK
CHO
XXXXXXXXXX XXXXXXXXXX
FIG. 4. Fluorogram of Man-6-P receptors immunoprecipitated
from the gradients in Fig. 3, showing the differences in the distri-
ution of receptors among Golgi subfractions in different cell types.
The fractions co
espond to those in Fig. 3. In Golgi subfractions
obtained from clone 9 cells, Man-6-P receptors are concentrated in
dense (cis) subfractions (lanes XXXXXXXXXXIn NRK cells they have a
oader 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