Growth, Differentiation and Sexuality

178 B.C.K. Lu
drial fission during cell death. Instead, yFis1p
limits mitochondrial fission and death by blocking
an irreversible step mediated by Dnm1p that leads
to loss of function of mitochondria (Fannjiang
et al. 2004). Interestingly, yFis1p has a duel
function. One works for mitochondrial fission
in normal growing cells, the other for regulating
the cell-death pathway in much the same way
as mammalian Bcl-2; over-expression of mammalian
Bcl-2 or Bcl-xL from a GAL1 expression
plasmid restores viability of acetic acid-treated
Δfis1 cells as efficiently as over-expressed yeast
yFis1. However, Bcl-2 or Bcl-xL fail to replace
the fission function of yFis1p (Fannjiang et al.
2004). Another gene has been identified in yeast
(YHR 155W) that codes for a mitochondrial
protein, Ysp1 (yeast suicide protein 1). This gene is
required for amiodarone-induced mitochondrial
fragmentation (also known as the thread-grain
transition), and it acts at a late stage, long after
ROS levels increase (Pozniakovsky et al. 2005).
Taken all together, these data led Bossy-Wetzel
et al. (2003) to conclude: “mitochondrial fission
per se does not result in cell death. However,
cell death does not occur without mitochondrial
fragmentation”.
The best evidence that links mitochondrial
fragmentation to cell death came from studies in
C. elegans. Here Drop1-induced mitochondrial fission
leading to PCD is egl-1- (egg laying-defective),
ced-4-, and ced-3- (cell death-defective) dependent;
the transcription activation of egl-1 is the earliest
event signaling commitment to the cell-death
pathway (Jagasla et al. 2005). In C. elegans development,
certain cells are destined to live and others
are destined to die, all in one and the same animal.
Jagasla et al. (2005) expressed a mitochondrial
matrix-targeted green fluorescent protein (mitoGFP)
under the control of the egl-1 promoter in
wild-type embryos. The appearance of GFP signal
marks the onset of apoptotic process, and this only
in cells destined to die. Mitrochondria are stained
with the fluorescent dye rhodamine B hexyl ester
and examined by confocal time-lapse microscopy.
Interestingly, within 8 min 52 s of transcription
activation, mitochondrial network in GFP-positive
cells starts to break down, resulting in a few
clusters of mitochondrial fragments located at the
periphery of the cells at 16 min 8 s. In the same
experiment with animals homozygous for the egl-1
loss-of-function mutation, cells destined to die do
not die, and mitochondria in GFP-positive cells
retain their tubular network. Thus, mitochondrial
fragmentation is an early event in the apoptotic
pathway. It should be noted that EGL-1 is also
a BH3-only protein, an activator of apoptosis,
and its effect on mitochondrial fragmentation is
specific for apoptosis (Jagasla et al. 2005).
VI. Autophagy and the Type II
Programmed Cell Death
A. Autophagy Is a Cellular Recycling Program
Proteins and cell organelles like ribosomes, mitochondria
and peroxisomes may be degraded for
nutrient reuse. This is distinct from the ubiquitinproteasome
pathway discussed in this chapter. Autophagy
is an inducible pathway; it can be triggeredbyacarbonoranitrogensourcestarvation
that inhibits TOR (target of rapamycin) serine/threonine
protein kinase, an upstream nutrient
sensor (Raught et al. 2001). Autophagy can
also be induced by a treatment with rapamycin.
The terminal target site of autophagic degradation
in yeasts, and probably in all fungi, is in the vacuole,
which is the equivalent of lysosomes of higher
eukaryotes. The loading of hydrolytic enzymes to
the vacuole/lysosome is achieved by three separate
pathways: (1) by trans-Golgi network (TGN), (2)
by the multi-vesicular body (MVB) pathway (Khalfan
and Klionsky 2002), and (3) by the cytoplasmto-vacuole
targeting (Cvt) pathway(Reggioriand
Klionsky 2002). The Cvt pathway is a biosynthetic
vacuolar trafficking pathway and it operates constitutively
under nutrient-rich physiological conditions,
but not induced by starvation. The formation
of Cvt vesicles shares the same processes as those
of autophagosomes.
There are twomajorcatabolicmulti-membrane
trafficking pathways in yeast: the macroautophagy
and the microautophagy pathways, and these are
inducible by nutrient starvation. Macroautophagy
is a generalized multi-membrane trafficking pathway
for all proteins and organelles. A large number
of genes involved in autophagy have been identified,
and these are named apg, aut, andcvt genes
(see the list in Klionsky and Emr 2000; Reggiori and
Klionsky 2002). The last step is to deliver all cargos
to the terminal acceptor compartment, the vacuole
(Huang and Klionsky 2002; Meiling-Wesse et al.
2002a,b; Mizushima et al. 2002; Wang et al. 2002).
Thesinglemembraneishydrolyzedbylipases,releasing
the cargos for hydrolytic degradation.