Unsolved Mystery

Autophagy: A Forty-Year Search for a Missing

Membrane Source
Gabor Juhasz*, Thomas P. Neufeld

A
utophagy is the major self-degradative process in case, making room and providing nutrients for the cells that
eukaryotic cells, with fundamental roles in cellular will eventually give rise to the adult insect [6]. In mammals,
and organismal homeostasis, and is involved in autophagy occurs in virtually all cells at a basal rate, and has
many developmental and pathological situations. Structures been shown to be required for the elimination of old and
targeted for autophagic destruction are sequestered nonfunctional organelles and protein complexes [7]. Growth-
into newly emerging double-membrane vesicles called promoting hormones such as insulin inhibit autophagy [8],
autophagosomes, and delivered for lysosomal degradation. whereas glucagon, synthesized in response to low blood
Despite recent advances in understanding the molecular sugar levels, induces this process [9]. Autophagy has been
mechanisms of autophagy, a long-standing question suggested to play a protective role during aging, cell death,
concerning the source of the autophagic membrane remains defense against intracellular pathogens, neurodegenerative
unresolved. Two major alternatives can be considered: the diseases, and tumorigenesis, emphasizing the biological and
membrane may be derived from a pre-existing cytoplasmic medical importance of autophagy [2,10].
organelle such as the endoplasmic reticulum (maturation
model), or assembled from constituents at its site of genesis The Morphology of Autophagy
(assembly model). During autophagy, large membrane-bound portions of
the cytoplasm are delivered for destruction to lysosomes,
Introduction organelles loaded with various acidic hydrolases specialized
The stability of all biological systems—from single cells to for rapid and effective degradation of cellular and
ecological communities—is based on the continuous turnover extracellular material. As early as the 1960s, the morphology
of individual units. Just as new organisms are born to replace of the major pathway of autophagy, macroautophagy
dying ones, turnover of constituents within a cell ensures that (simply referred to as autophagy hereafter), was established
old or damaged macromolecules and organelles are replaced by electron microscopic studies [1,9]. Upon induction of
by newly synthesized ones. This constant replacement autophagy, a membrane cisterna (fold of membrane) known
underlies the adaptability of biological systems, for example as the isolation membrane (IM; sometimes referred to as
allowing cells to rapidly change their metabolism in response phagophore in mammals [11]) appears and curves around
to a changing environment. Cellular homeostasis (the ability part of the cytoplasm. Sealing of the edges of the IM results
to maintain a stable condition inside the cell) is therefore in a unique double membrane vesicle, the autophagosome.
based on the proper balance of synthesis and destruction. Soon after forming, autophagosomes fuse with a lysosome,
In eukaryotic cells, various specialized cytoplasmic enzymes where degradation of the delivered material for recycling
are responsible for the specific degradation of proteins (the takes place (Figure 1A and 1B). The membranes of IMs and
ubiquitin-proteasome pathway), lipids, ribonucleic acids, autophagosomes differ from other membranes in the cell
sugars etc.; these degradation events can be crucial to the in having few intramembrane proteins, evident by electron
execution of cellular signaling and metabolic pathways. microscopy [12–14].
In contrast, nonspecific degradation of these materials The dynamic membrane rearrangements of autophagy
occurs through autophagy (“self-eating” in Greek; in this are also unique in that topologically intracellular material
case at a subcellular level) [1]. This process plays several (cytoplasm) becomes converted into topologically
important roles in the life of a cell. During times of starvation,
autophagy ensures survival by randomly degrading bulk Citation: Juhasz G, Neufeld TP (2006) Autophagy: A forty-year search for a missing
cytoplasm including organelles to provide breakdown membrane source. PLoS Biol 4(2): e36.
products that can be used for energy and synthetic processes
Copyright: © 2006 Juhasz and Neufeld. This is an open-access article distributed
[2]. In response to a change in available nutrients, autophagy under the terms of the Creative Commons Attribution License, which permits
is also used to specifically eliminate obsolete metabolic unrestricted use, distribution, and reproduction in ay medium, provided the original
author and source are credited.
organelles in several yeast species [3]. In multicellular
organisms, autophagy is tightly controlled through several Abbreviations: Atg, autophagy-related gene; ER, endoplasmic reticulum; IM,
signaling pathways [4,5], and has been integrated into isolation membrane; PAS, preautophagosomal structure
various developmental and physiological events, such as the Gabor Juhasz and Thomas P. Neufeld are in the Department of Genetics, Cell
remodeling of cells, tissues, organs, or even the entire body. Biology, and Development, University of Minnesota, Minneapolis, Minnesota, United
To take an extreme example, nearly all of the larval tissues of States of America

a metamorphosing insect are self-digested inside the pupal Competing Interests: The authors have declared that no competing interests exist.

* To whom correspondence should be addressed. E-mail: juhas001@umn.edu (GJ);

Unsolved Mysteries discuss a topic of biological importance that is poorly neufeld@med.umn.edu (TPN)
understood and in need of research attention.
DOI: 10.1371/journal.pbio.0040036

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 the IM forms upon induction of autophagy. Atg proteins
form three main multiprotein complexes: an autophagy-
specific phosphatidyl-inositol 3-kinase (PI3K) complex, the
Atg1 kinase complex, and two closely linked ubiquitin-like
protein conjugation systems. Current research is aimed at
understanding how these complexes interact to promote
autophagy.
Multicellular organisms harbor homologs of most of the
yeast Atg genes, reflecting the evolutionary conservation of
autophagy and its molecular components. There are also
some important differences. For example, IMs in yeast bud
from the single PAS, whereas in mammalian cells, IMs are
formed throughout the cytoplasm, and no definitive PAS is
observed. The presence of multiple homologs of some Atg
genes also suggests an as-yet unknown complexity of the
process in metazoans.
One of the ubiquitin-like proteins, Atg8 (called LC3
in mammals), appears to be associated with IMs and
autophagosomes through a lipid anchor [17], and therefore
represents the first known protein covalently attached to
these membranes. Atg8/LC3 fused to a fluorescent tag is now
commonly used as a light microscopic marker to follow the
formation of early autophagic structures (Figure 1C) [18].

Potential Membrane Sources of the IM

Despite the recent advancement of autophagy research,
one fundamental question remains unanswered: how does
the autophagic membrane form? The membranes of IMs
and autophagosomes are of the thin type (6–7 nm), like
the membranes of the endoplasmic reticulum (ER), cis-
DOI: 10.1371/journal.pbio.0040036.g001 Golgi, nuclear envelope, and inner and outer membranes
of mitochondria. In contrast, membranes of the plasma
Figure 1. The Dynamic Membrane Events Involved in Autophagy
membrane, lysosomes, and most of the Golgi are thick
(A) Upon induction of autophagy, a membrane sac called the isolation
membrane (IM) forms and engulfs portions of the cytoplasm. Sealing of (9–10 nm), due to their different lipid composition (e.g.,
its edges gives rise to the double-membrane bound autophagosome. more cholesterol) and higher protein content. A review
Fusion of the outer membrane with a lysosome results in formation of article written nearly 40 years ago by de Duve and Wattiaux
an autolysosome, in which the inner autophagosomal membrane and its [1] already mentioned that the source of the sequestering
contents are degraded.
(B) Starvation-induced autophagosomes (AP) and autolysosomes (AL) in membrane “has given rise to many speculations,” including
the fat body (the functional analogue of the liver) of a fruit fly larva. Note the ER, Golgi complex, and de novo formation. Below we
that APs contain intact cytoplasm, whereas the contents of ALs show consider the evidence relating to two general models of
various stages of degradation.
(C) Liver cells of starved mice carrying a fluorescently tagged LC3 autophagic membrane formation.
transgene, labeling cup-shaped and ring-shaped structures that
correspond to IMs and autophagosomes, respectively. The Maturation Model
Images courtesy of Ryan Scott (B) and Dr. Noboru Mizushima (C).
All cellular membrane is generally thought to derive from
the ER. Membrane from the ER is transported through the
extracellular (the lumen of lysosomes) without crossing a secretory pathway by a continuous cycle of vesicular budding
membrane barrier [15]. In contrast, material delivered to the and fusion. Based on these observations, one model posits
lysosome by other routes either remains extracellular, in the that the IM is derived from the ER or another pre-existing
case of endocytosis (formerly referred to as heterophagy [1]), organelle upon induction of autophagy. For example, a
or crosses a membrane, as in the case of lysosomal enzyme portion of the ER may become cleared of ribosomes and
synthesis. fold onto itself to form the IM (Figure 2A); alternatively,
vesicles may bud off from the ER and fuse together to form
Identification of Genes Involved in Autophagy the IM (Figure 2B). This theory is supported by the similar
Yeasts, like humans, alter their metabolism to meet the membrane thickness of these organelles (see earlier),
amount and type of available nutrients. And, unlike humans, by the putative identification of ER proteins in IMs and
they are much easier to study genetically. Little was known autophagosomes in mammalian cells [19] (but see also [20]
about the molecular mechanisms of autophagy until the last for an extended discussion of this point), and also by the
decade, when yeast genetic screens revealed ~27 autophagy- observation that IMs are often observed between parallel ER
related genes (Atg), whose products are necessary for cisternae in secretory cells [13]. In yeast, recent molecular
autophagy [16]. Most Atg proteins localize at least transiently genetic studies showed that genes necessary for ER trafficking
to a single well-defined spot in the cytoplasm known as the are required for autophagy [21]. These data are consistent
preautophagosomal structure (PAS), a site from which with a membrane contribution from the ER, although they

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 answer to this elusive question is thus likely to spring from new
technologies, such as improved in vivo lipid labeling methods
capable of specifically marking different cell membranes
in live cells [25]. In addition, the recent and ongoing
identification of the Atg proteins, many of which specifically
localize to the PAS and IM, provides a powerful new set of
tools to address this problem. This list is likely to grow as
additional factors are identified, perhaps based on their co-
localization with known Atg proteins, and through genetic
screens in other organisms. Among the most promising of
the known factors is Atg9, the sole known integral membrane
protein involved in IM formation. Atg9 cycles through the
PAS and other unidentified punctate structures [23,26].
DOI: 10.1371/journal.pbio.0040036.g002
As this protein is presumably membrane-bound from its
Figure 2. Possible Membrane Sources of the IM inception, live tracking of Atg9 is likely to identify at least
Some of the possible scenarios for IM formation are illustrated here. a subset of the contributing membrane. These reagents,
According to the maturation model, upon induction of autophagy, in conjunction with standard cell biology approaches, thus
membrane may be directly derived from the ER by folding (A), or in represent novel means of investigating the membrane source.
the form of vesicular transport (B). In the assembly model, membrane
may be assembled de novo at the site of IM formation, originating from For example, a specific pool of lipids or Atg proteins en
nonvesicular transport, such as micellar, as shown in (C), or local synthesis route to the forming and growing IM could be followed by
(D). See text for further details. photobleaching recovery or pulse-chase methods [27,28]. In
addition, these tools will allow researchers to delve further
may instead simply reflect a genetic requirement for one or into this mysterious puzzle, addressing a number of closely
more proteins synthesized in the ER. related fundamental questions: As new membrane is added
to the growing IM, is it recruited to the tips, center or entire
The Assembly Model surface? How is this membrane targeted to the IM? Does
Recent real-time studies have revealed that newly forming induction of autophagy stimulate membrane production,
IMs continue to elongate until the final sealing of the or are pre-existing stores of lipid sufficient? Are the known
autophagosome [22]. This observation is difficult to reconcile Atg proteins directly involved in membrane recruitment or
with the direct maturation of an existing organelle into an synthesis, and if so what are the mechanisms involved? A new
IM. An alternative model posits that the IM is assembled de generation of tools and a plate full of questions is attracting
novo. In this model, nonvesicular transport (Figure 2C) or new researchers to this expanding field, leading to an
local synthesis (Figure 2D) of lipids supplies the material for accelerated pace of discovery. Future work will provide plenty
the growing membrane. Currently, support for this model of new information to ingest.

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