exocitosis

Box 1 | Classification criteria for endocytic mechanisms in the membrane56, which defines a further locati
could recruit or trap specific membrane comp
Fission mechanism Finally, siRNA-mediated depletion of endogeno
Dynamin
CtBP3/BARS
tillin-1 in cells caused a marked decrease in dyn
Lipid-based independent endocytosis of fluid-phase m
GPI-APs and CtxB. This implicates flotillin-1
of the cytoplasmic components of CI endocy
However, it remains to be determined whether flo
has a direct role in cargo selection and vesicle bu
Lipid composition
Cholesterol or whether it is a cargo molecule that is recogn
Raft components the CI endocytic machinery.
GTPases
Sphingolipids
Cargo CDC42
Ligand or receptor ARF6 Mechanisms for membrane budding
Fluid RhoA The generation of endocytic carriers at the cell
GPI-AP involves membrane deformation, its growth
Adhesion molecules
Virus spherical bud or tubule and, finally, membrane s
Toxin In the clathrin-mediated pathway, many mem
Bacteria deforming proteins such as clathrin, epsin and dy
Coat components
Clathrin have been reported to be involved65. However,
Caveolin this well-studied pathway, the physico-chemical
Flotillin nism of membrane deformation and pinching are
Adaptors Actin cytoskeleton
Actin understood. Recently, two theoretical models hav
Regulation Actin-binding proteins proposed that consider the material characte
Kinases Actin-regulatory proteins
Phosphatases of the membrane and the machinery that is neces
Ubiquitylation the generation of membrane buds (BOX 3). Thes
GEFs, GAPs retical approaches are important because they p
Many different factors affect clathrin-independent (CI) endocytosis (see figure) and new ways of thinking about the budding proce
could potentially be used to enumerate and classify these uptake mechanisms. may rationalize the classification of these end
Alternatives to the endocytic mechanism classification system that is used in this pathways based on the actin regulatory GTPases
Review (FIG. 3) might include cholesterol dependence, actin sensitivity or association
with lipid rafts (which are often synonymous with detergent-resistant membranes Itineraries of CI endocytic cargo
(DRMs); see the list below). Another scheme is based on the pathways that different Extensive studies that detail the itineraries of v
viruses can use when they enter cells. cargo molecules have been carried out. For ex
• Cholesterol dependence. Most endocytic pathways are sensitive to cholesterol endocytosed GPI-APs can be detected in tubular i
perturbation, and clathrin-dependent92,93 and CI pathways29,48,49 are inhibited by the
ations that subsequently undergo fusion to ge
removal of cholesterol.
distinct early endosomal compartments, termed G
• Lipid rafts. DRM-associated proteins can be accommodated in almost all endocytic enriched early endosomal compartments (GEEC
pathways42.
CtxB is endocytosed via small tubular or ring-like
• Actin sensitivity. Many clathrin-dependent and -independent endocytic pathways known as clathrin- and dynamin-independent c
EVIEWS

Phagocytosis Macropinocytosis

Clathrin- Caveolin-
Particle
dependent dependent Clathrin- and caveolin-
endocytosis endocytosis independent pathways
Dynamin
Actin filaments
Clathrin Caveolin

CLIC

Uncoating
Caveosome
GEEC

Lysosome Early endosome

Figure 1 | Pathways of entry into cells. Large particles can be taken up by phagocytosis, whereas fluid uptake occurs by
macropinocytosis. Both processes appear to be triggered by and are dependent on actin-mediated remodelling of the
plasma membrane at a large scale. Compared with the other endocytic pathways, the size of the vesicles formed by
phagocytosis and macropinocytosis is much larger. Numerous cargoes (TABLE 1) can be endocytosed by mechanisms that are
independent of the coat protein clathrin and the fission GTPase, dynamin. This Review focuses on the clathrin-independent
pathways, some of which are also dynamin independent (FIGS 2,3). Most internalized cargoes are delivered to the early
endosome via vesicular (clathrin- or caveolin-coated vesicles) or tubular intermediates (known as clathrin- and dynamin-
independent carriers (CLICs)) that are derived from the plasma membrane. Some pathways may first traffic to intermediate
compartments, such as the caveosome or glycosyl phosphatidylinositol-anchored protein enriched early endosomal
compartments (GEEC), en route to the early endosome.

in our classification scheme is the requirement for Dynamin-dependent CI pathways. Caveolae-mediated

dynamin, thus, CI pathways are neatly divided in terms endocytosis is perhaps the best-characterized dynamin-
of those that use a dynamin-mediated scission mech- dependent CI pathway. Caveolae are 50–80 nm flask-
anism (dynamin-dependent), and those that require shaped plasma membrane invaginations that are marked
REVIEWS

v-ATPase growth factors, while inhibiting catabolic pathways,

SNAREs as autophagy through the phosphorylation of Unc
Ion channels, LAMPs Organelle
transporters fusion like kinase 1 (ULK1)10. Of note, mTORC1 also regu
H+ Tethering lysosome re-formation during autophagy, a pro
factors
pH 4.5–5.0 Organelle
that helps to restore a full complement of funct
contacts lysosomes during prolonged starvation11.
Small Activation of mTORC1 requires its dynamic rec
Cholesterol Hydrolases GTPases
transporters ment to the lysosomal surface, which is mediate
Enzyme activators/ the amino acid-dependent activation of heterodim
protective factors Motility
Motor RAG GTPases and their interaction with Ragulator7,12–17
Transport proteins adaptors RAG-dependent recruitment of mTORC1 to the
Signalling somal surface can also be induced by cholesterol thr
Sugar complexes Metabolic the involvement of cholesterol-binding Niemann–
transporters signalling type C1 protein (NPC1)18. In addition to mTORC1,
GTPases modulate the lysosomal recruitment of o
Nucleoside Transcription
transporters
Gene nutrient-responsive molecules, including the mTO
factors regulation
regulators tuberous sclerosis complex (TSC) 19
Amino acid folliculin (FLCN)–folliculin-interacting protein 1 (F
transporters complexes20, as well as TFEB21, a master modulat
Fig. 1 | Properties of lysosomes. The lysosome comprises a specific set of luminal, lysosome biogenesis and autophagy8 (see also the
integral-membrane and peripherally associated proteins. The lysosomal lumen contains tion Lysosomal adaptation) (FIG. 2). Additional de
acid hydrolases that degrade different substrates, enzyme activators and protective on lysosomal nutrient sensing and on the regulation
factors that aid in the degradation, as well as transport factors such as lipid transfer functions of mTORC1 at the lysosomal membrane c
Niemann–Pick type C2 protein (NPC2), which shuttles free cholesterol to NPC1 for found in other recent reviews9,17.
export from the lysosome. The acidic pH of the lysosomal lumen is maintained by a
vacuolar ATPase (v-ATPase) embedded in the limiting membrane. In addition, the Lysosomal Ca2+ signalling. Lysosomal Ca2+ is key for
lysosomal membrane comprises highly glycosylated lysosome-associated membrane ious lysosomal functions. Release of Ca2+ is require
proteins (LAMPs) that among other functions protect the lysosomal membrane from
the fusion of lysosomes with other cellular struct
degradation, ion channels and transporters that maintain ion homeostasis, cholesterol
and other lipid transporters, solute carriers that participate in the export of sugars, including endosomes, autophagosomes and the pl
nucleosides, amino acids and other products of lysosomal degradation, and SNAREs that membrane22,23, thereby regulating endocytic memb
mediate fusion of lysosomes with other organelles. The cytosolic face of lysosomes serves trafficking, autophagy and repair of membrane dam
as a platform for the dynamic association of proteins and protein complexes (shown in (see also the section Interactions with other organe
the right part of the figure), including the mechanistic target of rapamycin complex 1 and Furthermore, lysosomal Ca2+ release is involved in
its regulators that transduce nutrient and growth factor signals (FIG. 2), transcription formation of contact sites with the endoplasmic r
factors such as TFEB and TFE3 that regulate lysosome biogenesis, autophagy and energy ulum (ER), which in turn is able to refill the lysos
se: also involved in a positive-feedback loop with TFEB,

Calcium signalling
Lysosome re-formation
8
Dynamin

Lysosome
Autolysosome

Endosome–lysosome fusion

Lysosome Endosome
SNAREs

TFEB nuclear translocation Nucleus

P
TFEB TFEB TFEB

Calcineurin
 Phospholipid
Lipid transfer ER Lysosome positioning
and movement
3
ORP1 L
? 4
6
ORP5 Catch and release
2 RAB7
PtdIns4P Ub
Cholesterol VPS13C 5 USP1 5

NPC1 PtdIns3 P
RNF2 6
NPC2 TOLLIP p6 2
VAP
7
1
STARD3 Lysosome PtdIns3P
Protrudin
TBC1D15

FIS1 RAB34
RILP

Lysosomal
and 11 PtdIns(3,5)P2
mitochondrial Folliculin
Tether? TRPML1 8
dynamics
(regulation of Mitochondrion 10
fission) Ribosomal ANXA11 Golgi
protein 9 complex Perinuclear
Ca2+ SYT7
clustering
RNA PtdIns(4,5)P2
mRNA granule Peroxisome
Ribosomes

Localized Cholesterol
translation transfer

Fig. 5 | Membrane contacts between lysosomes and other organelles. Lysosomes establish versatile contacts with
various organelles. Several of these contacts now have well-established functional consequences. An important function
of contacts with the endoplasmic reticulum (ER) is lipid transfer, likely mediated by STARD3–VAP (step 1), ORP5–NPC1
(step 2), VPS13C–VAP (step 3) and/or RAB7–ORP1L–VAP (step 4) (of note, VAP exists as VAPA and VAPB paralogue with at
least partially redundant functions)127–129. STARD3, ORP5, Niemann–Pick type C1 protein (NPC1) and ORP1L serve as lipid
transfer proteins. VAPs are ER-resident proteins and, other than for their contribution to membrane contacts, their roles in
lipid transport processes are not clear. Lysosome–ER contacts also control lysosome positioning and movement. This can
occur by the dynamic attachment of lysosomes to perinuclear ER via the RNF26–p62–TOLLIP–USP15 system. Here,
lysosomes can be tethered to the ER via p62 in a manner that is regulated by the ubiquitylation status of p62, which is in
turn dynamically modulated by the opposing activities of the ubiquitin (Ub) ligase RNF26 and the deubiquitylating
enzyme USP15 (step 5)131. Proteins at lysosome–ER contacts also regulate coupling of lysosomes to cytoskeletal motors.
For example, ORP1L regulates the cholesterol-dependent coupling of lysosomes to dynein–dynactin via RAB7 and RILP
(step 6)130, whereas protrudin regulates the coupling of lysosomes to kinesin 1 via RAB7 and FYCO1 (step 7)154 (FIG. 6).
Contacts with the Golgi complex via RAB34, RILP and folliculin promote perinuclear clustering of lysosomes in response
to nutrient starvation (step 8)134. Contacts with peroxisomes via synaptotagmin 7 (SYT7) and phosphatidylinositol
4,5-bisphosphate (PtdIns(4,5)P2) mediate NPC1-dependent cholesterol transfer (step 9)133. Annexin 11 (ANXA11)-mediated
contacts promote the axonal transport of RNA granules for local translation of mRNAs encoding mitochondrial proteins
(step 10)140,141. Finally, lysosomes establish dynamic contacts with mitochondria, where tethering is promoted by RAB7 and
counteracted by FIS1, which inhibits RAB7 via the activity of the RAB7 GTPase-activating protein TBC1D15 (step 11)136.
For a more detailed description of these mechanisms, see REFS3,114,124. PtdIns3P, phosphatidylinositol 3-phosphate;
PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate.
 ↓ Amino acids
↓ Growth factors
Retrograde Alkalinization
Protein aggregates
Reactive oxygen species
Cholesterol accumulation

Lysosome
Ca2+
RAB7
TMEM55B ALG2 TRPML1 ORP1L
JIP4 RILP

Dynein Dynactin

– +

Microtubule
KIF5 KIF1
Kinesin 1 Kinesin 3
KLC ARL8
BORC
FYCO1
RAB7 SKIP

Ragulator

↑ Amino acids Anterograde

↑ Growth factors
Acidification
Oncogenesis

Fig. 6 | Machineries involved in lysosome motility. Several adaptor or scaffold complexes mediate coupling of lysosomes
to dynein–dynactin or kinesins (predominantly kinesin 1 (composed of two KIF5 heavy chains and two light chains (KLC),
and kinesin 3 (a homodimer of KIF1 heavy chains)) for retrograde and anterograde transport, respectively. These complexes
are sensitive to environmental or metabolic conditions, allowing control of lysosome motility and positioning in response
to different stimuli. The small GTPases RAB7 and ARL8) regulate lysosome motility and positioning in addition to lysosome
interactions with other organelles (FIG. 4). RAB7 regulates coupling of lysosomes to both dynein–dynactin (via RILP))
and kinesin 1 (via FYCO1)). ARL8 regulates coupling of lysosomes to kinesin 1 (via SKIP) and kinesin 3 (probably directly).
For a more detailed description of these mechanisms, see REFS142,228. BORC, BLOC1-related complex.

An additional system comprises the ER-anchored protein
protrudin, which binds to RAB7 and PtdIns3P on the
lysosomal membrane to promote the interaction of
kinesin 1 with another PtdIns3P-binding adaptor, FYCO1,
for movement of lysosomes towards the cell periphery154
(FIG. 6). Amino acid starvation reduces PtdIns3P levels,
dissociating protrudin and FYCO1 from lysosomes
and thus causing perinuclear clustering of lysosomes132.
knockdown of the same proteins or BORC, or knock-
out of the corresponding genes, reduced it132,149,156. Since
mTORC1 is activated by engagement of growth factor
receptors at the plasma membrane, these effects were
proposed to depend on the distance of lysosomes from
the plasma membrane — the assumption being that the
proximity of mTORC1 to the plasma membrane facili-
tates its activation. Other studies, however, showed
REVIEWS

Membrane expansion and shaping

Inducing
Cytoplasm
signals
Isolation membrane components
or phagophore

Membrane
nucleation

Pore
closure
Lysosome
Autolysosome Autophagosome

Mammal

Degradation

Yeast/plant

Vacuole

Degradation

Fig. 1 | Overview of the process of autophagic degradation. In both yeasts and mammals, autophagosome biogenesis
begins with the process of ‘membrane nucleation’ to generate the autophagosome precursor. Subsequently, the precursor
is transformed into the isolation membrane or phagophore, which expands and bends into a spherical shape. Finally, the
pore in the isolation membrane closes to complete the formation of the autophagosome. During this process, various
intracellular materials are sequestered into the autophagosome. To complete autophagy, the outer autophagosomal
 c Lipid flipping?

P PtdIns3P
Lipid Atg18/WIPI
transfer
Atg2/ATG2
Mammal
SEC23B-containing
COPII coat

Isolation
ERGIC membrane

d
es
PI3P-dependent
translocation

SEC12
ER exit sites ER
 lar importance. The possible curvature-stabilizing effect fission events (
of lipidated Atg8-family proteins as discussed above248 vesicle format
may also work to stabilize the isolation membrane edge membrane pore
(FIG. 5a); this idea is consistent with the results that the that ESCRT com
protein level and functionality of Atg8 correlate with brane and prom
autophagosome size197,200. It was reported
also involves the
(related to RAB
Mammal
and Atg17 (REF.2
ESCRT-I, ESCRT-III, VPS4 tory to the fact
(ESCRT-0, ESCRT-II?) autophagic bod
vesicles released
some–vacuole
Pore component ATP
Isolation
closure shaping system
Autophagosome
membrane isolation memb
ESCRT-III
Conclusions a
Identification o
S. cerevisiae molecular diss
Atg17, Vps21 (RAB5) Molecules that f
ESCRT-0, ESCRT-I, ESCRT-II,
ESCRT-III, Vps4 inently includin
way and memb
Fig. 6 | Mechanism of autophagosomal pore closure. ESCRT-III mediates pore closure and their memb
in the isolation membrane. In Saccharomyces cerevisiae, the other ESCRT (endosomal ferently involved
sorting complexes required for transport) components (ESCRT-0, ESCRT-I and ESCRT-II), these players ha
Vps4 and Vps21 (a RAB5 homologue) were also reported to be important for this process.
functions of m
In addition, the ESCRT-III component Snf7 interacts with Atg17, a possible mechanism
for the recruitment of the ESCRT machinery to the isolation membrane. In mammals, and how autoph
it remains elusive whether this process involves ESCRT-0 and ESCRT-II. There is also these proteins a
the site for auto