CBG.

01: Prokaryotes and

eukaryotes

An inside-out origin of the

EK cell
- models always assumed that the
nucleus and endomembrane
system evolved within the
cytoplasm of a PK cell
- a PK nucleus-like cell extruded
membrane-bound blebs beyond
its cell wall; these blebs
functioned to facilitate material
exchange with ectosymbiotic proto-
mitochondria;
- the cytoplasm was then formed through
the expansion of blebs around proto-
mitochondria, with continuous spaces
between the blebs giving rise to the
endoplasmic reticulum, which later
evolved into the EK secretory system
- further bleb-fusion steps yielded a
continuous plasma membrane which
served to isolate the endoplasmic
reticulum from the environment
- mitochondria are derived from
endosymbiotic alfa-proteobacteria via
phagocytosis

EXISTING THEORIES FOR THE ORIGIN OF

NUCLEUS: novel structure formed within the
boundaries of an existing, and largely unaltered,
plasma membrane (outside-in models)
result of sequential phagocytosis and
endosymbiosis
not clear whether mitochondria were acquired
before or after the nucleus
SYNTROPHIC CONSORTIUM MODEL: simultaneous
fusion of a symbiotic community composed of all
3 partners
ENDOSPORE MODEL: nucleus evolved when a Figure 1
cell enclosed its sister after cell division, similar Inside-out model for the evolution of eukaryotic
to the way in which endospores are formed in cell organization. Model showing the stepwise
certain Gram-positive bacteria
evolution of eukaryotic cell organization from (A) an
require supplementary explanations for the
eocyte ancestor with a single bounding membrane
origins of: endomembrane system, physical
continuity of inner and outer nuclear and a glycoprotein rich cell wall (S-layer) interacting
membranes, formation of nuclear pores with epibiotic -proteobacteria (proto-
EK genome likely represents a combination of 2 mitochondria). (B) We envision the eocyte cell
genomes: archaeal and proteobacterial forming protrusions, aided by protein-membrane
more recent phylogenetic data have suggested interactions at the protrusion neck. These
that mitochondria were present in the last EK
protrusions facilitated material exchange with proto-
common ancestor; this has lad to the formulation
of new autogenous models in which the mitochondria. (C) Selection for a greater area of
acquisition of mitochondria predates the contact between the symbionts would have led to
formation of nuclear compartment bleb enlargement and the eventual loss of the S-layer
from the protrusions. (D) Blebs would have then
been further stabilized by the development of a
symmetric nuclear pore outer ring complex (Figure 2)
and through the establishment of LINC complexes
that, following the gradual loss of the S-layer,
physically connected the original cell body (the
 nascent nuclear compartment) to the inner bleb
membranes. (E) With the expansion of blebs to
enclose the proto-mitochondria, a process that
would have facilitated the acquisition of bacterial
lipid biosynthesis machinery by the host, the site of
cell growth would have progressively shifted to the
cytoplasm, facilitated by the development of
regulated traffic through the nuclear pore. At the
same time, the spaces between blebs would have
enabled the gradual maturation of proteins secreted
into the environment via the perinuclear space
through glycosylation and proteolytic
cleavage. (F) Finally, bleb fusion would have
connected cytoplasmic compartments and driven the
formation of an intact plasma membrane, perhaps
through a process akin to phagocytosis whereby one
bleb enveloped the whole. This simple topological
transition would have isolated the endoplasmic
reticulum from the outside world, driven the full
development of a system of vesicular trafficking, and
established strict vertical transmission of
mitochondria, leading to a cell with modern
eukaryotic cell organization.
DE ROSS: the starting point was a proto-
eukaryote with a double membrane that secreted
membranous extracellular vesicles that fused to
form an enclosing plasma membrane
SEARCH: autonomy of nuclei in syncytia
Subcellular localization of protein N-
glycosylation
- extracellular protrusions arose to facilitate
material exchange with the external environment
- the starting point might have been a PK cell
similar to an eocyte: single lipid bilayer, simple
cell wall, relatively well-developed cytoskeleton
- many Archaea and eocytes exhibit protrusions to
increase the surface-to-volume ratio
- selective pressures for protrusion growth: an
increasingly intimate association with symbiotic
proto-mitochondria
- inside-out model is consistent with hydrogen
hypothesis, except that, by identifying eocytes
as the most likely host, a methanogenic host
metabolism seems unlikely
- mitochondria in modern EK retain close
metabolic, physical and regulatory linkages with
ER; the ER has been found to play a critical role
in mitochondrial fission
- the extent to which membrane protrusions
swelled beyond the S-layer would have
depended on the relative osmotic pressure of the
cell and its environment, and the sophistication
of osmoregulation
- the stabilization of blebs would have been
facilitates by the evolution of an outer ring of
nucleoporins supporting a second area of
positive curvature on the outside of the cell wall
- the majority of the structural lipids within EK cell
membranes are quite distinct from archaeal
lipids; in fact, they bear many similarities to
those found in bacteria; this strongly suggests
that EK acquired their bacterium-like lipids from
mitochondria; modern evidences:
o mitochondria retain a critical role in EK fatty
acid metabolism and in lipid synthesis,
generating many of their own lipids
(cardiolipin)
o ER is the major site of lipid and membrane
synthesis in modern EK, with many of the
enzymes involved found concentrated at the
ER-mitochondrial contact sites
o connections between ER and mitochondria
remain important sites of lipid traffic in
modern EK
o inositol lipids (ubiquitous in EK, but a tiny
fraction of the total lipids in membranes), are
common in EK and archaea, but not bacteria
- so, phagocytosis is a phenomenon that appeared
after the acquisition of mitochondria because
archaeal membranes are not likely to go through
the changes needed for engulfment (they are too
rigid)
- cellular and nuclear divisions: at first, the nuclear
membrane would have been continued with the
ER, so the division of the cell is the same as the
division of the nucleus; in modern cells, there is
open and closed mitosis, depending on the
integrity of the nuclear membrane;
Model for the evolution of cell division. Cell
division is depicted for the ancestral eocyte (A), and
at two intermediate stages in the evolution of
eukaryotes, before (B) or after (C)bleb fusion.
Following the acquisition of blebs, ESCRTIII is used to
drive the scission of cytoplasmic bridges connecting
cells (likely aided by the archaeal-derived actin
cytoskeleton [51]), while LINC complexes and the
formation of new nuclear pores restore cell and
nuclear organization following division. Mitochondrial
segregation is likely aided by host induced Dynamin-
mediated scission within the endoplasmic reticulum
(not depicted), as observed in modern eukaryotes

- if the LCA of all EK lacked a cell wall, its easy to
understand why different EK phyla acquired
biochemically distinct cell walls
- regulated secretion and vesicle trafficking drove
elaboration of the cytoplasmic compartment
- the presence of a plasma membrane:
o the cell has tighter control over material
exchange with the environment, preventing
direct diffusion into and out of the ER space
o ensures the vertical inheritance of
mitochondria, facilitating its coevolution with
the host
- the order in which the secretory and endosomal
The stepwise evolution of eukaryotic vesicle
trafficking. From left to right the figure depicts a
simple hypothesis for the evolution of the eukaryotic
secretion and vesicle trafficking systems. Initially,
proteins (black dots) would have been secreted from
ribosomes bound to rough endoplasmic reticulum
(ER) into the space at the bases of blebs by the Sec
translocase and signal recognition particles (SRP)
[50]. Secreted proteins could then undergo stepwise
processing using machinery adapted from that used
to process glycoproteins in the archaeal S-layer (that
is, through N-linked glycosylation of asparagine-X-
serine or asparagine-X-threonine-containing proteins,
and proteolysis [99]). The elaboration of ER tubules
and local membrane bending regulated by the Sar1
GTPase, in the presence of generic SNAP Receptors
(SNAREs) (blue bars), would have enabled the
transient fusion of ER to the outer cell membrane,
releasing these glycosylated proteins into the
extracellular space. These transient openings would
have been closed by Dynamin-mediated fission.
Specialized SNARE proteins (differently colored bars)
and Dynamin (triple diagonal lines), would then have
generated vesicular intermediates to better regulate
secretion. The intercalation of additional processing
steps and the diversification of these protein families
would have yielded compartment-specific paralogs,
together with the evolution of regulatory Arf and Rab
GTPases, and a Golgi compartment. Finally,
membrane bending machinery together with
Dynamin, actin, and Rho family GTPases would have
been co-opted to drive endocytosis, phagocytosis,
and the development of the modern retrograde
trafficking pathway
trafficking systems evolved is almost precisely
opposite under inside-out and outside-in models
- the machinery for generating cilia might have
similarities to that generating protrusions early in
the evolution of EK
- this proposes a new model for nuclear interphase
pore insertion: the first step in the generation of
a new nuclear pore is recruitment of elements of
the outer ring of the NPC to bend the inner
nuclear membrane outwards
- cells with long lived nuclear blebs will tend to be
those characterised by low rates of compartment
fusion, as might be indicated by their having ER
and Golgi that lack fenestrae
The principle of parsimony states that we should
favour models that explain observations while
drawing on the fewest ad hoc assumptions. The
inside-out model is parsimonious in that, simply by
assuming that the cytoplasm was formed from
extracellular blebs, it can explain diverse features of
modern eukaryotic cell organization. For example the
model explains the existence of a nuclear
compartment that lacks any internal membrane-
bound organelles, why typical eukaryotic cells are
much larger than most prokaryotes yet have a
nucleus similar in size to many eocytes, why there is
a double nuclear membrane whose periplasmic
space is continuous with ER, why protein N-
glycosylation is initiated in the nuclear envelope, and
why the ER tends to be continuous with the nuclear
envelope. Outside-in models account for the nuclear-
ER connection, but new assumptions need to be
added to account for ER continuity, for the lack of
similarity between the S-layer and eukaryotic cell
walls, for N-linked glycosylation machinery having
been relocalized from the plasma membrane (S-
layer) to the ER, for the fusion of vesicles to form a
single nucleus that lacks functional ribosomes, and
so on. While these data come far short of ruling out
the outside-in view, we believe that the inside-out
model offers a simpler explanation for these data.
The second class of evidence comprises assorted
quirky features of eukaryotes that would not be
predicted under conventional models for the origin
of eukaryotes, yet seem to arise quite simply from
the inside-out model. Thus, the inside-out model
naturally explains why ER tends to show high levels
of continuity, even in multinucleate syncytia, where
nuclei achieve high degrees of autonomy. Likewise,
the model explains the close functional connections
between ER and mitochondria and the important
roles both organelles play in lipid synthesis. And, as
one final example, the inside-out model provides a
logical explanation for the presence of
phosphoinositides in the nucleus and their role in
regulating mRNA processing.
The third class of evidence involves inferences drawn
from phylogenetic analyses of eukaryotic gene
families. Phylogenetic analysis of the Ras GTPase
superfamily has been used to argue that secretion
and exocytosis evolved before endocytosis (this
conclusion is, however, uncertain given the
phylogenetic tree obtained). Such an order of
evolution is predicted by the inside-out model, not - first articulated in 1910 by the Russian botanist
the outside-in model. Stronger evidence in support of
our theory arises from phylogenomic studies that
identify -proteobacteria, and hence mitochondria,
as the source of eukaryotic lipid biosynthesis and
transport genes. This conclusion is further bolstered
by phylogenetic results supporting the eocyte
hypothesis because this tree topology makes it even
less probable that eukaryotic lipids were vertically
inherited from the last common ancestor of Bacteria
and Eukarya. If bacterial lipids were acquired from
mitochondria, then it seems almost certain that
phagocytosis could not have been the means by
which a close symbiosis was established between
mitochondria and their eukaryotic hosts. This follows
because the acquisition of bacterial lipids would have
been a pre-requisite for the dynamic changes in
membrane structure required for phagocytosis. The
inside-out model is unique among competing
theories in allowing close symbiosis of mitochondria
and their eukaryotic hosts to be established without
phagocytosis, minimizing the number of membrane-
interacting proteins affected by a changeover in
membrane chemistry. Thus, phylogenetic analysis of
lipid biosynthesis genes provides compelling support
for the inside-out model over alternatives that rely on
one or more early phagocytic events.
In addition to the diverse lines of evidence that are
compatible with the inside-out theory, it is worth
highlighting that the inside-out model involves a
simple series of steps, each of which draws upon
plausible ecological and selective drivers. In the early
stages, selection favors closer and more extensive
physical contact between the host and its
ectosymbiotic proto-mitochondria to improve
material exchange. Bleb growth and the elaboration
of the ER would then have been favored by
protecting the mitochondrial flora from being lost to
the environment and by protecting ectosymbionts
from parasites. In addition, elaboration of the ER
would have facilitated the development of a much
more sophisticated secretory system that allowed the
stepwise processing of proteins prior to export.
Movement of mitochondria into the cytoplasm would
have eliminated an intervening membrane, further
enhancing metabolic exchange. And, finally,
formation of a complete plasma membrane would
have been advantageous because it closed-off the ER
from environmental challenges, such as pathogenic
bacteria or viruses, and enabled the complete control
of protein modification during secretion.
Symbiogenesis
Konstantin Mereschkowski and advanced and
substantiated with microbiological evidence by
Lynn Margulis in 1967
- EK organelles are thought to represent formerly
free-living PK taken one inside the other in
endosymbiosis around 1.5 billion years ago:
o mitochondria developed from proteobacteria:
Rickettsiales
o chloroplasts developed from cyanobacteria
(nitrogen-fixing filamentous cyanobacteria)
CHRISTIAN
DE DUVE:
peroxisomes
hydrophobicity hypothesis: highly hydrophobic
proteins are not easily transported through the
cytosol and therefore these proteins must be
encoded in their respective organelles
code disparity hypothesis: the limit on transfer
is due to differing genetic codes and RNA editing
between the organelles and the nucleus
redox control hypothesis: genes encoding
redox reaction proteins are retained in order to

might have been the

first endosymbionts,
allowing the cell to
whitstand growing
amounts of free
molecular oxygen in the
Earths
atmosphere
it now appears that they may be formed
de novo, contradicting the
idea that they
have a symbiotic
origin
- it is considered to be a
type of saltational
evolution

KEELING &ARCHIBALD: the

usual way to distinguish
organelles from endosymbionts
is by their reduced
genome sizes
- as an endosymbiont evolves into an organelle,
most of their genes are transferred to the host
cells genome
- there are 3 main possible fates for genes over
evolutionary time:
o loss of functionally redundant genes, in which
genes that are already represented in the
nucleus are eventually lost
o transfer of genes to the nucleus: nuclear
genes have expended and became more
complex; many nuclear genes originating from
endosymbionts have acquired novel functions
unrelated to their organelles
cDNA hypothesis: use of mRNA to transport effectively couple the need for repair and the
genes from organelles to the nucleus where they synthesis of these proteins
are converted to cDNA and incorporated into the the assembly of membrane proteins,
genome (study of genomes of flowering plants); particularly those involved in redox reactions,
later, it was observed that cDNA is more likely to requires coordinated synthesis and assembly of
recombine with its native mitochondria DNA than subunits; however, translation and protein
with the hosts genome transport coordination is more difficult to control
bulk flow hypothesis: escaped DNA is the in the cytoplasm
mechanism of gene transfer: disturbances to
organelles (autophagy, gametogenesis, cell ROBERTO CAZZOLLA GATTI: endogenosymbiosis:
stress) release DNA which is imported into the not only organelles endosymbiotic, but pieces of
nucleus and incorporated into the nuclear DNA genetic material from endosymbiotic parasites
using non-homologous end joining; (gene carriers such as viruses, retroviruses and
bacteriophages) are included in the hosts
o endosymbiont genes remain in the organelles: nuclear DNA, changing the hosts gene
plastids and mitochondria retain genes expression and contributing to the process of
encoding rRNA, tRNA, proteins involved in speciation
redox reactions, proteins required for
transcription, translation and replication
EVIDENCE THAT MITOCHONDRIA AND PLASTIDS
AROSE FROM BACTERIA (WIKI):
New mitochondria and plastids are formed only
through binary fission, the form of cell division used
by bacteria and archaea.
If a cell's mitochondria or chloroplasts are
removed, the cell does not have the means to
create new ones. For example, in some algae, such
as Euglena, the plastids can be destroyed by
certain chemicals or prolonged absence of light
without otherwise affecting the cell. In such a case,
the plastids will not regenerate.
Transport proteins called porins are found in the
outer membranes of mitochondria and chloroplasts
and are also found in bacterial cell membranes
A membrane lipid cardiolipin is exclusively found
in the inner mitochondrial membrane and bacterial
cell membranes.
Some mitochondria and some plastids contain
single circular DNA molecules that are similar to the
DNA of bacteria both in size and structure.
Genome comparisons suggest a close
relationship between mitochondria and Rickettsial
bacteria.
Genome comparisons suggest a close
relationship between plastids and cyanobacteria.
Many genes in the genomes of mitochondria
and chloroplasts have been lost or transferred to the
nucleus of the host cell. Consequently, the
chromosomes of many eukaryotes contain genes
that originated from the genomes of mitochondria
and plastids.
Mitochondrial and plastid ribosomes are more
similar to those of bacteria (70S) than those of
eukaryotes.
Proteins created by mitochondria and
chloroplasts use N-formylmethionine as the
initiating amino acid, as do proteins created by
bacteria but not proteins created by eukaryotic
nuclear genes or archaea
Protein misfolding and
degenerative diseases
- 1917: Hermann Staudinger proposed that
proteins are macromolecules made of
small-molecule constituents linked
together by chemical bonds
ANFINSEN(1972): all the information needed for
the protein folding is found in the AA sequence;
denatured protein using urea (disrupts
noncovalent bonds) and mercaptoethanol
(reduces disulphide bonds); the structures
refolded spontaneously into the original form
which minimizes the overall free energy of the
protein
LEVINTHALs paradox: a protein folds rapidly
because its AA interact locally, thus limiting the
conformational spacethat the protein has to
explore and forcing the protein to follow a funnel-
like energy landscape that allows it to fold into
the most stable configuration possible
- big proteins are sometimes helped by
chaperones to be folded properly;
ROM LASKEY (1978) nucleoplasmin acted as
a chaperone, accompanying and supervising the
activity of the histones
- the existence of chaperones implies that
some proteins have inherently unstable
conformations, being able to fold in
alternatively stable, but useless/toxic
ways
AMYLOID DEPOSITS: misfolded proteins are
often insoluble and tend to form long linear and
fibrillar aggregates
- this conformational change is more likely
to occur in proteins that have repetitive
amino acid motifs (polyglutamine
Huntingtons disease)
INFECTING CONFORMATIONS: often, the toxic
proteins are capable to interact with other native
copies of the same protein, catalysing their
transition in the toxic state;
- many proteins move from alfa helix to
beta sheet;
- misfolded proteins cause diseases =
prions (Creutzfeldt-Jakob, kuru) and can
be transmitted between species; they also
occur naturally in unicellular organisms
(yeast)
- protein aggregation diseases are not
exclusive to the central nervous system;
they can also appear in periphereal
tissues; in general, the genes and protein
products involved in these kinds of
diseases are called amyloidogenic
Disease
Alzheimer's disease
Parkinson's disease
Parkinson's disease
Parkinson's disease
 Parkinson's disease
Parkinson's disease
Parkinson's disease
Parkinson's disease
Amyotrophic lateral sclerosis
Huntington's disease
LAURENCE A. MORAN
- the pure form of the tree of life was now
abandoned and replaced with a net of life
concept
CARL WOOSE: (1) archebacteria form a
monophyletic group, (2) this clade is sufficiently
different from all other PK to deserve elevation to
a separate domain called Archaea (the other 2
domains are Bacteria and Eukarya), (3) EK are
more closely related to archaebacterial than to
other PK, (4) the root of the universal tree of life
lies in the branch leading to Bacteria
- growing recognition that SSU- based (sequence
of the small ribosomal RNA subunit) trees are not
as reliable as we once thought
- ribosomal RNA trees are prone to long branch
attraction artifacts to a greater extend than trees
based on AA sequences
- sometimes, there occurs lateral/horizontal gene
transfer, but: which genes were transferred and
which ones represent the true species
phylogeny?
- other way of explaining the conflict is to invoke
whole genome fusions followed by selective loss
of half the genes;
NORMAN PACE: the right gene for analyses: (1)
the gene must be universal (2) the gene must
have resisted lateral gene transfer (3) the gene
must be large enough to provide useful
phylogenetic information
LAURENCE A. MORAN (added criteria): (4) the
gene must be unique, or if it isnt, paralogues
must be easily recognized (5) the gene must
encode a protein because its much more
accurate to analyse AA sequences than nuclei
acid sequence (6) the gene must be highly
conserved in order to retain significant sequence
similarity at the deepest level
at those criteria, ribosomal RNA doesnt do so
well
- part of the problem in using rRNA sequences in
deep phylogenies is that they are too divergent
- the phylogeny of ATPase was one of the
strongest bits of evidence for the Three Domain
Hypothesis back in 1989 but further work has
shown that these genes (proteins) now refute the
hypothesis;
- Hsp70 is the main chaperone in all species: it is
responsible for the correct folding of proteins as
they are synthesised
- diversity among bacteria seems to be ancient
- all trees have potential problems of saturation
and long branch attraction
complexity hypothesis: there is a core of genes
that have never been transferred from one
species to another because they are all part of a
large complex; they represent the true
phylogeny of a lineage and all other genes have
been acquired later; this true core is made of
genes that are involved in translation;
FORD DOOLITTLE, contra arguments:
some key translation components do not
agree with the rRNA tree, refuting the idea that
all genes of the complex evolved together
parts of rRNA that interact with ribosomal
proteins are highly conserved so there is very
little difference between species; the parts that
dont interact are the ones that are variable;
lateral gene transfer of rRNA genes from one
species to another wouldnt have much effect
since the only parts that differ are the parts that
arent necessary
it does not mean that the genes werent
transferred, but that theyve always been
transferred together
 he proposes a Darwinian threshold: after this
We have, for several decades, thought that our moment organisms can be related as a tree; the
job was to uncover the structure of a Tree of Life, first to pass the threshold were bacteria and then
whose reality we did not question. But really, archaebacteria
what we have been doing is testing Darwins
hypothesis that a tree is the appropriate
representation of lifes history, back to the
beginning. Like any hypothesis, it could be false. THE PHYLOGENY TREE (UCLA.BERKLEY)
CARL WOESE: discovered Archaebacteria and - domains: Bacteria, Archaea, Eukaryota: no one of
proposed the 3 domain hypothesis that not only these groups is ancestral to the others, and each
claims that archaebacteria are a domain, but also shares certain features with the others as well as
claims that EK descend from a primitive having unique characteristics of its own
achaebacterium
primitive life existed as a community of cells
- it appears that the most of the biological
that freely exchanged genes; they shared a basic diversity of EK lies among protists, and many
translation system for making proteins, but had scientists feel it is just as inappropriate to lump
little else in common all protists into a kingdom as it was to group all
PK