Biomedical Engineering Reference
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S. cerevisiae
compared to other organisms such as Pichia pastoris, since the
former seems to lack a system that assembles exit sites together at the level
of the ER (Rossanese
et al.,
1999). However, despite these differences, the
basic process of secretion is highly conserved between
S. cerevisiae
and
higher eukaryotic cells (Kaiser and Huffaker, 1992).
The Sec7-1 mutant was isolated in the original selection for secretory
pathway mutants (sec mutants) in
S. cerevisiae
(Novick
et al.,
1981; Novick
et
al.,
1980). In
Sec7-1
cells maintained at restrictive temperature, there is a
block in the secretory pathway at the level of exit from the Golgi apparatus
(Franzusoff and Schekman, 1989; Julius
et al.,
1984; Nishikawa
et al.,
1990;
Novick
et al.,
1981; Stevens
et al.,
1982). Rambourg, Clermont and Képès
carried out a detailed study of the evolution of Golgi elements in the
Sec7
-
I
mutant held at restrictive temperature (Rambourg
et al.,
1993b). Cells were
prepared using the Karnovsky procedure consisting of impregnation with a
1 : 1 mixture of osmium tetroxide and potassium ferrocyanide. This technique
allows visualization of glycosylated membrane proteins, with a gradient of
coloration from the ER (faintly stained) to the late Golgi/plasma membrane
(darkly stained).The stereoscopic techniques based on thick section electron
microscopy that had been developed by Rambourg and Clermont were
adapted to the yeast system to examine the three-dimensional structure of
intracellular organelles. Over a time course of incubation of the
Sec7-1
mutant at 37
o
C, the Golgi tubular networks were progressively transformed
into stacks of saccules whose superposed elements were interconnected
along a
cis
-
trans
axis to form a membrane structure that is anatomically con-
tinuous. This process can be divided into several steps. First, after only seven
minutes at 37
o
C, one or more of the numerous Golgi tubular elements
became more extensive, with tubules accumulating in a network with the
same type of three-dimensional organization as in the wild type situation.
These networks were intensely stained, correlating well with the fact that
transport of secretory proteins is blocked at the exit of the Golgi, after the
Golgi glycosylation enzymes have acted. Very few ER-Golgi vesicles were
seen at this time point, indicating that as in the wild type, they continue to
be consumed rapidly at the
cis
side of the Golgi. After 15 minutes at restric-
tive temperature, the large, darkly stained tubular networks had become
aligned in a parallel fashion. Some cells had accumulated highly fenestrated
cisternae, suggesting that the tubular elements, once aligned in parallel, had
begun to undergo filling in of the tubular mesh to form fenestrated saccules.
After 30-60 minutes, only a few Golgi elements per cell were seen, and all
were composed of five to eight stacked, poorly fenestrated saccules. Hence
the process of membrane transformation from tubulation through fenestra-
tion to poorly fenestrated saccules had gone essentially to completion. Inter-
estingly, even after longer periods of incubation at the nonpermissive
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