Lennarz W.J., Lane M.D. (eds.) Encyclopedia of Biological Chemistry. Four-Volume Set . V. 4

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in viral infection and GCN2 by uncharged tRNAs in

amino acid starvation. Our group noted the existence of

a predicted transmembrane protein in the C. elegans

genome, with an N-terminal “extracellular” domain

similar to IRE1 and an intracellular domain similar to

PKR and GCN2, known eIF2

kinases. This protein,

which is conserved in metazoans (and absent from yeast)

and to which we gave the name PERK (PKR-like ER

Kinase), couples ER stress to eIF2

phosphorylation

and is essential to translational regulation by ER stress.

Cells lacking PERK are hypersensitive to ER stress

and in mammals, PERK activity is especially important

for preservation of cells engaged in high levels of

secretion. Thus humans with PERK mutations and

mice lacking the gene, develop diabetes mellitus and

skeletal defects attributed to dysfunction of insulin-

and collagen-secreting cells in the endocrine pancreas

and bone, respectively.

GENE EXPRESSION, EIF2

PHOSPHORYLATION AND INTEGRATION

SIGNALS IN THE UPR,

AND OTHER STRESS PATHWAYS

In addition to limiting the load of client protein on the

stressed ER (by inhibiting translation of most mRNA),

eIF2

phosphorylation paradoxically activates the

translation of the mRNA encoding the transcription

factor ATF4 (and likely that of other regulatory

proteins which remain to be discovered, Figure 3).

PERK thus plays an important role in up-regulating

target gene expression in the UPR. Expression proﬁling

of ER stressed wild-type and PERK 2 /2 cells reveals a

broad role for PERK in UPR-target gene expression,

with mild impairment in most target genes and severe

impairment in a smaller group. Among the UPR target

genes that are strongly impaired in PERK 2 /2 cells are

genes involved in amino acid transport and metabolism

and a gene, GADD34, which encodes a phosphatase

that dephosphorylates eIF2

and terminates signaling

by PERK. The aforementioned strong PERK target

genes play an important role in maintaining translation,

by providing building blocks for secreted protein

synthesis and by promoting eIF2

dephosphorylation.

Thus, PERK and eIF2

phosphorylation appear to have

a dual role in the UPR. Early in the response they

protect the stressed cells from ER overload whereas

later they promote conditions required for synthesis of

secreted proteins.

A considerable overlap exists between genes activated

by ER stress and genes activated by other stressful

conditions that promote eIF2

phosphorylation.

Thus, eIF2

phosphorylation integrates signaling in

several stress pathways. The extent of this integrated

stress response is not fully known, but it appears to

control important aspects of cellular and organismal

metabolism.

FIGURE 2 ATF6 activation by regulated intramembrane proteolysis. Membrane tethering inactivates the transcription factor ATF6, whose

effector domain is prevented from accessing its target genes in the nucleus. BiP binding to the lumenal domain of ATF6 retains the protein in the ER

compartment. However, under conditions of ER stress, BiP dissociates from ATF6, which then migrates (presumably by vesicular transport) to a

post-ER compartment that contains the proteases that liberate the effector domain to signal in the UPR.

UNFOLDED PROTEIN RESPONSES 323

A Common Mechanism

for Sensing ER Stress

The identiﬁcation of three different stress sensors in the

UPR has allowed a comparison between their mode of

activation. As mentioned earlier, overexpression of BiP

and other ER chaperones had been noted to repress

signaling in the UPR. In the equilibrated ER, all three

stress transducers, IRE1, ATF6, and PERK, are found in

a simple complex with BiP, which binds on the lumenal

side to their stress-sensing domains. These complexes

are rapidly dissociated under conditions of ER stress.

Furthermore, dissociation precedes activation of the

stress transducers. BiP dissociation promotes oligomer-

ization of IRE1 and PERK, which in turns leads to

transautophosphorylation and activation of down-

stream signaling. It seems likely that BiP-dissociation

under conditions of ER stress unmasks a homo-

oligomerization domain in the similarly structured

lumenal domains of IRE1 and PERK.

The chain of events in the case of ATF6 activation is

more complex, but conceptually similar. BiP binding

retains ATF6 in the ER, segregated from the proteases

that release its transcription factor portion, as these are

localized to a post-ER compartment. BiP dissociation

liberates ATF6 to migrate to the protease-containing

compartment, a migration that presumably takes place

by vesicular transport. Thus, dispensible molecules of

BiP, which are not engaged in chaperoning ER client

proteins actively repress signaling by all three known

transducers of the UPR. It would thus appear that the

recognition of unfolded and malfolded proteins is

relegated to professional chaperones and the stress

transducers passively monitor the degree to which the

latter are engaged by their clients. This arrangement of

the upstream steps of the UPR mirrors that of the

cytoplasmic heat shock response in which unfolded

proteins in the cytoplasm activate genes implicated in

folding and degrading cytoplasmic proteins.

FURTHER READING

Cox, J. S., and Walter, P. (1996). A novel mechanism for regulating

activity of a transcription factor that controls the unfolded protein

response. Cell 87, 391–404.

Harding, H., Zhang, Y., and Ron, D. (1999). Translation and protein

folding are coupled by an endoplasmic reticulum resident kinase.

Nature 397, 271–274.

Harding, H. P., Calfon, M., Urano, F., Novoa, I., and Ron, D. (2002).

Transcriptional and translational control in the mammalian

unfolded protein response. Annu.Rev.CellDev.Biol.18,

575–599.

Kaufman, R. J. (1999). Stress signaling from the lumen of the

endoplasmic reticulum: Coordination of gene transcriptional and

translational controls. Genes Dev. 13, 1211–1233.

Sidrauski, C., and Walter, P. (1997). The transmembrane kinase Ire1p

is a site-speciﬁc endonuclease that initiates mRNA splicing in the

unfolded protein response. Cell 90, 1031–1039.

Yoshida, H., Okada, T., Haze, K., Yanagi, H., Yura, T., Negishi, M.,

and Mori, K. (2000). ATF6 activated by proteolysis binds in the

presence of NF-Y (CBF) directly to the cis-acting element

responsible for the mammalian unfolded protein response. Mol.

Cell Biol. 20, 6755–6767.

BIOGRAPHY

David Ron obtained his Medical Doctorate at the Technion in Haifa,

Israel. Following residencies in internal medicine and endocrinology at

Mount Sinai Hospital and the Massachusetts General Hospital and a

research fellowship at Harvard Medical School, he took a faculty

position at New York University School of Medicine, where he is

currently a Professor of Medicine and Cell Biology. His laboratory

studies stress responses in eukaryotic cells, with a special emphasis on

signaling between organelles and the nucleus.

UNFOLDED PROTEIN RESPONSES 325

Urea Cycle, Inborn Defects of

Marsha K. Fearing and Vivian E. Shih

Harvard Medical School, Boston, Massachusetts, USA

Surplus nitrogen generated by amino acid metabolism

cannot be stored in mammals and must be excreted. The

urea cycle is the primary pathway for the disposal of

ammonium nitrogen by conversion of the toxic ammonia

molecule into innocuous urea. Inborn errors of metabolism

in the urea cycle can result in severe diseases in humans.

An accumulation of ammonia can lead to severe morbidity,

including brain damage and death.

Overview

The urea cycle is the major pathway for the disposal of

nitrogen in humans (Figure 1). Ninety percent of

ingested protein is metabolized to urea and excreted

through the urine. Ammonia is derived from a variety of

precursor protein sources. Part of the urea cycle resides

in mitochondria where ammonia is converted to

carbamoyl phosphate by carbamoyl phosphate syn-

thetase (CPS) along with its allosteric activator,

N-acetylglutamate. The next step involves carbamoyl

phosphate forming citrulline when condensed with

ornithine. This process is mediated by the enzyme

ornithine transcarbamoylase (OTC). Citrulline exits

the mitochondria and condenses with aspartate to

produce argininosuccinate. This compound is then

cleaved to arginine and fumarate by argininosuccinate

lyase. Arginine is hydrolyzed by arginase, thus releasing

urea and regenerating ornithine. Ornithine is then

shuttled into mitochondria by its own transporter.

Within the urea cycle ornithine is a conserved moiety,

it is neither formed nor lost. One atom of waste

nitrogen from ammonia and one from aspartate forms

a molecule of urea.

Liver is the only organ that contains the complete

urea cycle and is the major site of ureagenesis. Intestinal

epithelial cells have CPS and OTC for citrulline synthesis

and are the source of the circulating citrulline in plasma

that serves as a precursor for synthesis of arginine, a

semi-essential amino acid, in brain and kidney. These

organs have negligible arginase activity, hence they do

not produce urea.

Clinical Diseases

UREA CYCLE DEFECTS

Gene mutations resulting in urea cycle defects have been

described for each of the ﬁve urea cycle enzymes.

The individual disease states are N-acetylglutamate

synthetase (NAGS) deﬁciency, carbamoyl phosphate

synthetase (CPS) deﬁciency, ornithine transcarbamoy-

lase (OTC) deﬁciency, argininosuccinate synthetase (AS)

deﬁciency or citrullinemia, argininosuccinate lyase (AL)

deﬁciency or argininosuccinic aciduria, and arginase

(A1) deﬁciency.

The clinical features of the urea cycle disorders are

similar. Depending on the enzyme involved and the

residual enzyme activity, metabolic blocks in the urea

cycle will cause a resultant elevation of the plasma

ammonia level. Hyperammonemia is associated with

many of the clinical features, which include respiratory

alkalosis, seizures, acute encephalopathy, coma, and

even death. Spasticity, resembling cerebral palsy can be a

late complication. The most severe presentation is coma

in the neonatal period, but symptoms can occur later in

childhood as well. Metabolic stress, intercurrent illness,

or fever can result in endogenous protein catabolism

and precipitate a hyperammonemic crisis. In milder

cases, abnormal neuropsychiatric behavior can be the

only manifestation. Short, friable hair (trichorrhexis

nodosa) and liver ﬁbrosis are unique to argininosucci-

nate lyase deﬁciency.

The exact pathophysiology of hyperammonemia is

not known and the etiology of brain edema is still

unclear. Accumulation of ammonia and astrocytic glu-

tamine are likely to be contributory to the brain damage.

Except for the X-linked ornithine transcarbamoylase

deﬁciency, the enzyme defects of the urea cycle disorders

are inherited as autosomal recessive traits.

The gene for OTC is located on chromosome Xp2.1.

Thus, males with OTC deﬁciency are usually more

severely affected with neonatal onset of hyperammone-

mia, whereas the clinical presentation in females with

OTC deﬁciency can be quite variable, depending on

random X-chromosome inactivation (Lyon hypothesis).

OTHER HYPERAMMONEMIA STATES DUE

TO AMINO ACID TRANSPORTER DEFECTS

Amino acid transporter defects are disorders of com-

partmentation. The urea cycle enzymes are available

but the substrates are not transported to the proper

site, resulting in a functional enzyme deﬁciency.

Triple H Syndrome (Hyperornithinemia,

Hyperammonemia, Homocitrullinuria)

The HHH syndrome is a defect in ornithine mitochon-

drial transport, which prevents the recycling of

ornithine as the substrate for OTC. The end result

is a functional OTC deﬁciency and reduced urea

synthesis. These children present with episodic hyper-

ammonemia, ataxia, vomiting, irritability, lethargy,

and coma. They often come to attention due to

mental retardation and developmental delay, with

occasional seizure activity. The diagnostic markers

are a combination of high plasma ornithine, high

blood ammonia, increased urine orotic acid, and

high urine homocitrulline, the origin of which is

unclear. The treatment is similar to that for OTC

deﬁciency.

Lysinuric Protein Intolerance

Lysinuric protein intolerance (LPI), also known as

hyperdibasic aminoaciduria, is an autosomal recessive

disease with an extremely high prevalence in the Finnish

population. The gene mutation has been identiﬁed on

chromosome 14q12. LPI occurs from defects in the

cellular basolateral membrane transporter of the dibasic

amino acids, in renal tubules and the intestinal system.

This results in excessive renal loss of lysine, arginine, and

ornithine in the urine and poor gastrointestinal absorp-

tion of the same amino acids. Arginine and ornithine

deﬁciency impairs the function of the urea cycle and

results in hyperammonemia after protein intake. Symp-

toms of LPI include failure to thrive, vomiting, and

chronic lysine deﬁciency, which causes growth failure

with decreased bone density resulting in, osteoporosis.

LPI patients also have recurrent autoimmune deﬁciency

and infections due to low white and red blood cell

counts. Other organ involvement includes interstitial

lung disease and renal failure.

Citrin Deﬁciency (Citrullinemia Type II)

Citrullinemia type II was originally reported as an adult-

onset citrullinemia, mainly in Japanese patients, with

Carbamoyl

phosphate

N-acetyl

glutamate

Carbamoyl phosphate

synthetase

Ornithine

transcarbamoylase

Argininosuccinate synthetase

Argininosuccinate lyase

Arginase

Glutamate

N-acetylglutamate

synthase

Citrulline

Argininosuccinate

Arginine

Ornithine

Acetyl CoA

Urea

cycle

TCA

cycle

Urea

Mitochondrion

Cytosol

FIGURE 1 The urea cycle. (1) N-acetylglutamate synthetase, (2) carbamoyl phosphate synthetase, (3) ornithine transcarbamylase, (4)

argininosuccinate synthetase, (5) argininosuccinate lyase, (6) arginase, (7) mitochondrial ornithine transporter, and (8) ornithine aminotransferase.

NAG ¼ N-acetylglutamate, an allosteric activator of carbamoyl phosphate synthetase.

UREA CYCLE, INBORN DEFECTS OF 327

TABLE I

Diagnostic Characteristics of Urea Cycle Disorders and Hyperammonemic Syndromes due to Amino Acid Transporter Defects

Disorder Plasma Cit Plasma Arg Urine orotic acid

Other diagnostic

amino acids

Genetic

markers Enzyme assay Prenatal diagnosis

N-acetyl-glutamate

synthetase deﬁciency

# N AR; 17q21.31 Liver DNA testing

Carbamoyl phosphate

synthetase deﬁciency

## NAR;

chromosome 2p

Liver Linkage analysis; direct

mutational analysis

Ornithine

transcarbamoylase

deﬁciency

## " X linked; Xp2.1 Liver Molecular diagnosis

Argininosuccinate

synthetase deﬁciency

h #" AR;

chromosome 9q34

Liver; ﬁbroblasts;

lymphoblasts,

chorionic villi

Enzyme assay; direct mutational

analysis; Metabolite

measurement in amniotic ﬂuid

Argininosuccinate

lyase deﬁciency

"# ""Arginino-succinic acid

(blood and urine)

AR;

chromosome 7q

Liver; ﬁbroblasts,

red blood cells

Metabolite measurement in

amniotic ﬂuid

Arginase deﬁciency N "" AR;

chromosome 6q23

Liver, red

blood cells

Enzyme assay, DNA analysis

Lysinuric protein

intolerance

## ""arginine, lysine,

ornithine (urine)

AR;

chromosome

14q11.2

Triple H syndrome ## ""ornithine (blood)

" homocitrulline (urine)

AR;

chromosome

13q14

Fibroblasts,

Amniocytes

Enzyme assay in fetal blood,

DNA analysis

Citrin deﬁciency "# ""citrulline

(blood and urine)

AR;

chromosome

7q21.3

Liver DNA analysis

a liver-speciﬁc argininosuccinate synthetase deﬁciency.

The clinical presentation depends on the degree of

hyperammonemia and includes a sudden disturbance of

consciousness, restlessness, drowsiness, and coma with

cerebral edema. Recent studies have identiﬁed a

deﬁciency of the citrin protein, which is a mitochondrial

transporter of aspartate and/glutamate, as the under-

lying cause. This has been conﬁrmed by mutation

analysis. Citrin deﬁciency has now been identiﬁed in

citrullinemic patients of different ethnic backgrounds

with neonatal intrahepatic cholestasis and other forms

of severe hepatic dysfunction.

Diagnosis

All disorders have hyperammonemia. Plasma and urine

amino acid analysis reveal the speciﬁc diagnostic

patterns (Table I). The amino acids proximal to the

enzyme block are increased and those distal to the block

are decreased. When there is a blockage in the urea cycle

beyond the step of CPS, the carbamoyl phosphate

synthesized cannot be used for urea synthesis and is

shunted to the biosynthetic pathway of pyrimidine to

form orotic acid. Thus, orotic acid is increased in all urea

cycle disorders except for NAGS deﬁciency and CPS

deﬁciency. Diagnosis can be conﬁrmed by enzyme assay

in red blood cells (arginase deﬁciency) and cultured

ﬁbroblasts (AS deﬁciency, AL deﬁciency). Measurement

of CPS and OTC activities can only be done in liver

biopsy. Enzymatic details can be seen in Table II.

Alternatively, molecular DNA mutation analysis can

be useful for diagnosis as well as for future genetic

counseling.

For all of the urea cycle defects, except NAGS,

prenatal diagnosis is available. AS and AL deﬁciencies

are noted by ﬁnding increased citrulline and increased

ASA in amniotic ﬂuid. Amniocytes do not have all of the

urea cycle enzymes; only AS and AL are expressed. Fetal

liver biopsy is required for prenatal diagnosis of CPS and

OTC deﬁciencies, and fetal blood is needed for prenatal

diagnosis of arginase deﬁciency. For prenatal diagnosis

of HHH syndrome, the ornithine incorporation assay

can be performed in cultured amniocytes. In cases of

known mutations in a family, DNA analysis is the

preferred procedure.

Treatment

LONG-TERM MANAGEMENT

Reduction in protein intake while maintaining nutri-

tional needs to ensure growth and development remains

the mainstay of therapy. The goal of therapy is to reduce

the ﬂow of nitrogen for disposal through the urea cycle.

This can be achieved by restriction of dietary protein

intake by providing an alternative pathway for nitrogen

disposal.

Alternative pathway therapy for nitrogen excretion

diverts ammonia clearance through compounds other

than urea. Sodium benzoate is one of the drugs used

for this purpose. When administered it conjugates with

the amino acid glycine to form hippurate, which has

high renal clearance. One mol of nitrogen is excreted

per one mole of hippurate. Following the same

principle but with greater efﬁciency, sodium phenyla-

cetate conjugates with the amino acid glutamine to

form phenylacetylglutamine, which eliminates two

moles of nitrogen.

To maintain normal growth it is important to provide

enough essential amino acids by supplementing the diet.

In patients with a urea cycle defect, arginine synthesis is

reduced and arginine becomes a semi-essential amino

acid. Patients can be supplemented with citrulline, the

precursor of arginine, to restore total body arginine

pools or with arginine directly in patients with AS and

AL deﬁciency.

In addition to medical management, liver transplan-

tation has been used with some success for treatment of

these disorders.

TABLE II

Urea Cycle Enzyme Characteristics and Locations

Enzyme Cellular location Relative activity in liver pH optimum Tissue distribution

NAGS Mitochondrial matrix 0.01 8.5 L, I, (K)

CPS1 Mitochondrial matrix 3.1 6.8–7.6 L, I, (K)

OTC Mitochondrial matrix 73 7.7 L, I, (K)

AS Cytosol 1 8.7 L, K, F, (B)

AL Cytosol 2.4 7.5 L, K, F, (B)

A1 Cytosol 962 9.5 L, RBC, (K), (B)

B ¼ brain; I ¼ small intestine; K ¼ kidney; L ¼ liver; RBC ¼ red blood cells.

UREA CYCLE, INBORN DEFECTS OF 329

ACUTE MANAGEMENT

Hyperammonemic crisis is often precipitated by stressful

situations such as fever, trauma, infection, surgery, and

dietary indiscretion. The goal of treatment is to lower

the blood ammonia levels to prevent brain edema. The

most effective treatment is hemodialysis or peritoneal

dialysis. Nitrogen scavenger medications, such as

sodium phenylacetate and sodium benzoate, and argi-

nine are also given intravenously. Protein intake is halted

and an intravenous ﬂuid with a high dextrose (10%)

load is provided immediately to minimize catabolism

and therefore the source of ammonia. Intercurrent

infection should be treated appropriately.

Future Horizons

Future treatment modalities may include total or partial

organ transplant, stem cell transplant, and enzyme

replacement. Early diagnosis utilizing newborn screen-

ing and prenatal diagnosis will allow early treatment

and improve the outcome.

FURTHER READING

Berry, G., and Steiner, R. (2001). Long-term management of patients

with urea cycle disorders. J. Pediatrics 138(Suppl. 1), S56–S61.

Feillet, F., and Leonard, J. V. (1998). Alternative pathway therapy for

urea cycle disorders. J. Inher. Metab. Dis. 21(Suppl. 1), 101 –111.

Fernandes, J., Saudubray, J. M., and Van den Berghe, G. (eds.) (2000).

Inborn Metabolic Diseases: Diagnosis and Treatment, 3rd edition.

Springer, Berlin.

Morizono, H., Caldovic, L., Shi, D., and Tuchmon, D. (2004).

Mammalian N-acetylglutamate synthase. Mol. Genet. Metab. 81,

4–11.

Nyhan, W. L., and Ozand, P. T. (eds.) (1998). Atlas of Metabolic

Diseases. Chapman and Hall Medical, London.

Steiner, R., and Cederbaum, S. (2001). Laboratory evaluation of urea

cycle disorders. J. Pediatrics 138(Suppl. 1), S21–S29.

Summar, M., and Tuchman, M. (2001). Proceedings of a consensus

conference for the management of patients with urea cycle

disorders. J. Pediatrics 138(Suppl. 1), S6–S10.

BIOGRAPHY

Marsha K. Fearing is a Clinical Fellow at the Harvard Medical School

Genetics Training Program, specializing in Biochemical Genetics. She is

a member of the Scholars in Clinical Science Program at Harvard

Medical School. Her area of research is translational and public health

research in metabolism. Dr. Fearing is a member of Alpha Omega

Alpha, the American Academy of Pediatrics, and a candidate fellow of

the American College of Medical Genetics.

Vivian E. Shih is Professor of Neurology at Harvard Medical School,

Associate Neurologist and Pediatrician at Massachusetts General

Hospital, and Director of the Clinical Neurochemistry and Amino Acid

Disorders Laboratory at Massachusetts General Hospital. Her area of

research is inborn errors of amino acid and organic acid metabolism.

330 UREA CYCLE, INBORN DEFECTS OF

Vacuoles

Christopher J. Stefan and Scott D. Emr

University of California, San Diego, La Jolla, California, USA

Vacuoles and their mammalian counterparts, lysosomes, are

membrane-bound cytoplasmic organelles that contain an

assortment of soluble acid-dependent hydrolases and a set of

highly glycosylated integral membrane proteins. Most notably,

this organelle is an important site for the degradation of

cellular lipids, membrane-associated proteins, and cytoplasmic

proteins. In addition to its degradative functions, the vacuo-

le/lysosome plays an important role in pH and ion homeostasis

and is a site for the generation/storage of nutrients, amino

acids, antigens, and additional signaling factors.

Identiﬁcation/Discovery of

Vacuoles and Vacuolar Constituents

Vacuoles/lysosomes were ﬁrst visualized by independent

cell morphological studies undertaken in the 1950s by

the Palade and Novikoff laboratories. These studies

revealed that lysosomes appear as organelles with

0.5 mm diameter and a heterogeneous morphology,

containing electron-dense cores and, in some cases,

membrane vesicles. In the yeast Saccharomyces cerevi-

siae, vacuoles are larger with respect to cell size, but

display similar characteristics in terms of electron

density and intralumenal vesicles, when observed

under certain conditions (Figures 1A and 1B).

Biochemical studies performed by de Duve and

colleagues initially identiﬁed lysosomes as organelles of

highly buoyant centrifugal properties that contained a

number of hydrolytic activities. The hydrolases within

this fraction demonstrated attenuated activity depen-

dent on the integrity of their surrounding membrane.

Speciﬁcally, hydrolase activity was retained within the

limiting vacuole/lysosome membrane, restricted from

the cell lysate unless the limiting membrane was

disrupted. Since vacuoles/lysosomes are morphologi-

cally heterogeneous, biochemical studies have produced

the current deﬁnition of vacuoles/lysosomes as mem-

brane-bound acidic organelles that contain mature

acid-dependent hydrolases and certain integral mem-

brane-glycoproteins. Vacuoles lack other proteins that

distinguish them from endosomes (compartments that

deliver material to vacuoles), such as biosynthetic

sorting receptors and recycling cell-surface receptors.

As already mentioned, vacuoles/lysosomes are mem-

brane compartments that serve as a major degradative

compartment in eukaryotic cells. Both endogenous

and exogenous molecules can be delivered to vacuoles

through biosynthetic and endocytic pathways. In addi-

tion, vacuoles can degrade proteins transported from the

cytosol. The degradative function of these organelles

is carried out by numerous acid-dependent hydrolases

(e.g., proteases, lipases, glycosidases) contained within

its lumen (Figure 2). The outer, limiting membrane of

vacuoles also contains a set of highly glycosylated,

vacuolar/lysosomal-associated membrane proteins.

A subset exhibits known hydrolase activities (Figure 2),

while the functions of many are still unclear. Additional

vacuolar membrane proteins mediate transport of ions,

amino acids, and other solutes across the vacuolar

membrane and maintain an acidic lumenal pH in the

range of 4.5–6.5 (Figure 2).

Vacuole Function in Yeast

The vacuole of the yeast S. cerevisiae plays an important

role in protein degradation, pH regulation, ion homeo-

stasis, and nutrient storage. The yeast vacuole contains a

large number of hydrolases (proteases, lipases, glycosi-

dases; see Figure 2) with various substrate speciﬁcities.

Thus, it is a major site for degradation of cellular

proteins, lipids, and even whole organelles.

A remarkable feature of vacuoles is their acidiﬁca-

tion. In yeast, a speciﬁc vacuolar proton pump ATPase

(V-ATPase) mediates this process. Biochemical and

genetic approaches have identiﬁed 14 subunits of the

V-ATPase (encoded by the VMA genes for vacuolar

membrane ATPase). The V-ATPase subunit proteins

form two complexes, the peripheral V

subcomplex,

responsible for ATP hydrolysis, and the V

subcomplex,

responsible for proton translocation across the mem-

brane (Figure 2). Cells lacking a functional V-ATPase are

viable, but require an intact endocytic pathway to the

vacuole. Moreover, they fail to grow on media with

neutral pH, indicating that vacuole acidiﬁcation is an

essential feature. Consistent with this, the electrochemi-

cal gradient generated by the V-ATPase is necessary to

drive several other transport systems described in this

article (Figure 2).

In addition to its hydrolytic functions, the vacuole

serves as a storage compartment for several ions and

nutrients (Figure 2). For example, the Vcx1 protein acts

as a high capacity H

/Ca

2þ

exchanger in vitro and has

been implicated in the regulation of vacuolar calcium

ﬂux in vivo (Figure 2). The vacuole also stores both

phosphate and polyphosphate (Figure 2). Since hydroly-

sis of phosphate leads to the release of protons, a role for

polyphosphate in pH homeostasis has been suggested.

Polyphosphate is proposed to function as a cation

counter ion as well. Some amino acids are also stored

inside the vacuole at high concentrations. For example,

arginine accumulates in vacuoles at concentrations

nearly an order of magnitude greater than in the

surrounding media (Figure 2). Consistent with this,

several H

/amino acid anti-porter systems have been

identiﬁed in yeast. Pools of amino acids stored in the

vacuole may be utilized during nitrogen starvation.

Vacuole Biogenesis and Transport

Pathways to the Vacuole in Yeast

To carry out the numerous functions that vacuoles

perform, several transport pathways have evolved that

deliver cellular components to vacuoles. Model genetic

systems such as the yeast S. cerevisiae have been

instrumental in identifying both proteins and lipids

that constitute components of vacuoles and the machin-

ery involved in transport to vacuoles. Importantly, many

of these factors and transport pathways are conserved in

mammalian cells.

At least eight pathways have been identiﬁed in

vacuole biogenesis and protein transport to vacuoles in

FIGURE 2 Schematic overview of vacuolar components. The

vacuole lumen possesses several hydrolase activities and accumulates

ions, such as phosphate (PO

) and polyphosphates. The vacuolar

membrane also contains known hydrolases, such as ALP, and

proteases, such as dipeptidyl-aminopeptidase B (DPAP B). The

vacuolar ATPase (V

) functions as the primary proton pump that

generates an electrochemical gradient used to drive other vacuole

membrane transport systems, such as arginine (Arg) and divalent

cation (Ca

2þ

) anti-transporters.

FIGURE 1 Ultrastructural analysis of vacuole morphology in wild

type (A) and hydrolase deﬁcient (B) yeast cells reveals electron dense

cores and intralumenal membrane vesicles, respectively. (A) Wild type

cells were grown to mid-log phase, ﬁxed, and visualized by electron

microscopy. Vacuoles appear as dark, electron dense organelles due

to the accumulation of cellular proteins, lipids, and other molecules.

(B) In cells lacking vacuolar ATPase activity (vma4D mutant cells),

vacuoles appear translucent by electron microscopy and contain

intralumenal vesicles. Inset: High magniﬁcation image of vacuolar

vesicles (arrowheads) (v, vacuoles; n, nucleus; m, mitochondria;

bars ¼ 0.1 mm). (Reprinted with permission from Wurmser, A. E.,

and Emr, S. D. (1998). Phosphoinositide signaling and turnover:

PtdIns(3)P, a regulator of membrane trafﬁc, is transported to the

vacuole and degraded by a process that requires lumenal vacuolar

hydrolase activities. The EMBO Journal 17, 4930– 4942, copyright

1998 Oxford University Press.)

332 VACUOLES