Hermansky-Pudlak Syndrome: Vesicle Formation from Yeast to Man

Review

Hermansky–Pudlak Syndrome: Vesicle Formation from Yeast to Man

MARJAN HUIZING*1, RAYMOND E. BOISSY2 and WILLIAM A. GAHL1

1Section on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health,

Bethesda, MD; 2Department of Dermatology, University of Cincinnati, Cincinnati, OH, USA

*Address reprint requests to Marjan Huizing, Medical Genetics Branch, NHGRI, 10 Center Drive, MSC 1851, Building 10, Room 10C)103B,NHGRI, NIH, Bethesda, MD 20892-1851, USA. E-mail: [email protected]

Received 12 August 2002; in final form 14 September 2002

The disorders known as Hermansky–Pudlak syndrome (HPS)

are a group of genetic diseases resulting from abnormal

formation of intracellular vesicles. In HPS, dysfunction of

melanosomes results in oculocutaneous albinism, and absence

of platelet dense bodies causes a bleeding diathesis. In addition,

some HPS patients suffer granulomatous colitis or fatal

pulmonary fibrosis, perhaps due to mistrafficking of a subset

of lysosomes. The impaired function of specific organelles

indicates that the causative genes encode proteins operative in

the formation of certain vesicles. Four such genes, HPS1,

ADTB3A, HPS3, and HPS4, are associated with the four

known subtypes of HPS, i.e. HPS-1, HPS-2, HPS-3, and

HPS-4. ADTB3A codes for the b3A subunit of adaptor

complex-3, known to assist in vesicle formation from the trans-

Golgi network or late endosome. However, the functions of the

HPS1, HPS3, and HPS4 gene products remain unknown.

These three genes arose with the evolution of mammals and

have no homologs in yeast, reflecting their specialized function.

In contrast, all four known HPS-causing genes have homologs

in mice, a species with 14 different models of HPS, i.e.

hypopigmentation and a platelet storage pool deficiency.

Pursuit of the mechanism of mammalian vesicle formation

and trafficking, impaired in HPS, relies upon investigation of

these mouse models as well as studies of protein complexes

involved in yeast vacuole formation.

Key words: Yeast vacuole, Drosophila pigment granule,

Lysosome biogenesis, Protein trafficking, Oculocutaneous

albinism, Dense body, Vesicle

INTRODUCTION

Individual cellular functions rely upon specific, suitableenvironments provided by specialized compartments called

vesicles. Knowledge of the formation and maintenance ofvesicular compartments remains incomplete, but enormousadvances have been made in recent years. Some of our

new insights arose from investigations of yeast, flies,mice and men whose systems for vesicle formationand trafficking have gone awry. In humans, many of

these disorders are called, collectively, Hermansky–Pudlaksyndrome (1) or HPS. In this review, we describe theroles that the causative genes and their respective proteinshave played in studies of the genesis of intracellular

organelles.

The specific organelles affected in HPS are the melano-some, the platelet dense body, and the lysosome, which all

share certain integral membrane proteins. For example, themelanosome ME491, a melanoma-associated antigenexpressed strongly during the early stages of tumor progres-

sion, corresponds to the dense body CD63 or granulophysin,which is the same as the lysosome Lysosomal IntegralMembrane Protein-1 (LIMP-1) or Lysosomal Associated

Membrane Protein-3 (LAMP-3) (2). In HPS, dysfunctionof each organelle results in particular aspects of the disorder(3–8).

Impairment of melanosome formation causes different

degrees of albinism. Early in development, this leads to

Abbreviations – HPS, Hermansky–Pudlak syndrome; MRP, mannose-6-phosphate receptor; CPY, carboxypeptidase Y; ALP, alkaline phosphatase;PVC, prevacuolar compartment; MVB, multiple vesicular body; EEA1, Early endosome antigen 1; BLOC-1, Biogenesis of lysosome-related organellescomplex 1

PIGMENT CELL RES 15: 405–419. 2002 Copyright � Pigment Cell Res 2002

Printed in UK—all rights reserved ISSN 0893-5785

Pigment Cell Res. 15, 2002 405

improper migration of neural crest cells and abnormaldecussation of the optic nerve fibers. Dysfunction of retinalpigment epithelial cells, which among a host of other functions

provide nutritional support to rods and cones, may contributeto visual impairment. Visual acuity varies from 20 of 50 to 20 of400 in HPS (5, 9, 10), and is largely uncorrectable by refractivelenses. Patients nearly always have congenital horizontal

nystagmus, and strabismus is common (9, 10). On examina-tion, the irides transilluminate and the fundi exhibit scatteredhypopigmentation. Hair color ranges from dark brown to

completely white, and skin pigment also varies widely (11). Sunexposure predisposes to actinic keratoses and skin cancer,including basal cell carcinoma, squamous cell carcinoma, and

melanoma. Sun avoidance and the use of sunscreen areimportant therapeutic maneuvers in this regard.

The absence of platelet dense bodies, which are detectable on

whole mount electron microscopy (12), leads to a conditionknown as storage pool deficiency. Dense bodies containcalcium (which confers the electron density), polyphosphates,serotonin, adenosine triphosphate, and adenosine diphos-

phate (ADP). When stimulated, the dense granules releasetheir contents, and ADP triggers aggregation of neighboringplatelets. This secondary aggregation response, which can be

measured in a clinical laboratory, is impaired in HPS. Affectedindividuals have varying degrees of soft tissue and mucusmembrane bleeding (5). Bruises generally appear at first

ambulation, cuts bleed longer than expected, and epistaxis iscommon, especially before adolescence. Wisdom toothremoval and childbirth often bring the bleeding diathesis torecognition. Treatment involves local use of thrombin and

gelfoam, prophylactic use of 1-desamino-8-d-arginine vaso-pressin and, in serious cases, platelet transfusions.

Lysosomal involvement in HPS probably has several path-

ologic consequences. On a cellular level, some HPS patientsaccumulate ceroid lipofuscin, an amorphous lipid–proteincomplex, and this appears to be stored within lysosomes (13). It

accumulates within the kidney, bone marrow, spleen, liver,large intestine and sloughed urinary epithelial cells. In addi-tion, a subset of HPS patients develop pulmonary fibrosis

(14–16) and ⁄ or granulomatous colitis (5, 17, 18). As theseinvolve cells that lack melanosomes and dense bodies,lysosomal abnormalities are considered responsible. Thepulmonary fibrosis generally manifests in the fourth or fifth

decades of life and leads to death within 10 yr. The granulo-matous colitis of HPS presents, on average, at 16 yr of age (5)and resembles Crohn’s colitis in symptoms and response to

therapy.The specialized cell, tissue, and organ systems of Homo

sapiens manifest specific signs and symptoms caused by

vesicular abnormalities. Some of the proteins aberrant inhumans with HPS may have progenitors in other, moreprimitive species. We now trace the progression of vesicle-

forming systems from yeast to Drosophila to mouse and man.

Yeast

Yeast genetics, studied largely in Saccharomyces cerevisiae,has made the greatest impact on our knowledge of the

molecular mechanisms involved in targeting of proteinsto lysosomes. The yeast vesicle corresponding to the

mammalian lysosome is the acidic vacuole, which plays acentral role in yeast physiology. The vacuole influences pHand osmoregulation, protein degradation, and storage of

amino acids, small ions, and polyphosphates. Molecularmachineries and biosynthetic pathways involved in theformation of the yeast vacuole and mammalian lysosomeare remarkably conserved (19–21). Many newly synthesized

proteins reach the vacuole after being diverted at the lateGolgi complex to endosomal compartments (22).

Genetic screens have identified a large number of yeast

mutants that are blocked at various points along thispathway. Some mutants mislocalize yeast vacuolar proteins(vps, for vacuolar protein sorting deficient) (23–26), and

others exhibit decreased vacuolar protease activity (pep, forpeptidase deficient) (27). Genetic, biochemical and cellbiological studies on these mutants have provided molecular

insights into the various pathways for delivering proteins tothe yeast vacuole.

The biosynthetic sorting of proteins from the late Golgicomplex to the yeast vacuole involves transport along two

distinct routes the �carboxypeptidase Y� (CPY) pathway andthe �alkaline phosphatase� (ALP) pathway (Fig. 1). Mostvacuolar proteins travel through the CPY pathway, from the

late Golgi through a prevacuolar compartment (PVC) to thevacuole (25, 28). This pathway resembles mannose-6-phos-phate receptor (MPR)-mediated sorting in mammalian cells

(19, 21). The ALP pathway sorts proteins from the Golgicomplex directly to the vacuole, without passage through aprevacuolar compartment (25, 28).

The CPY pathwayCarboxypeptidase Y, a soluble hydrolase within the vacuolarlumen, serves as a prototype for the CPY pathway (Fig. 1A).

This pathway requires the functional products of more than100 genes. Mutations in any of these genes result in missortingof proteins to the cell surface by a default pathway and, in some

cases, abnormal vacuolar morphology (25, 26).The CPY pathway can be divided into four stages. The first

stage involves protein sorting into membranes ⁄ vesicles at the

late Golgi, and transport of these vesicles toward the PVC.Proteins functioning in this stage include clathrin, theadaptor complex 1 (AP-1), vps10p (the CPY receptor),Peptidase deficient-1 (PEP1) (identical to vps10), drs2 (a P-

type adenosine triphosphatase), and all �Class D� vpsproteins, such as vps1p (a dynamin homolog), vps15p(homologous to the Ser ⁄ Thr family of protein kinases),

vps34p (a PI3-kinase homolog), vps8p (which interacts withvps21p), vps21p [a rab ⁄ ypt small guanosine triphosphatase(GTPase)], vps9p (a guanine nucleotide exchange factor),

and vps45p (a Sec1p family member regulating certain (targetmembrane)-soluble N-ethylmaleimide-sensitive factor attach-ment protein receptor (t-SNARES)) (25, 26, 29, 30).

The second stage of the CPY pathway consists offormation and maturation of the PVC, which is identical tothe mammalian multiple vesicular body (MVB). Mutants ofall �Class E� vps proteins, including vps4p, vps27p, vps28p,

result in PVCs having a multilamellar structure. Thismembrane accumulation appears to result from a block inmembrane trafficking out of the PVC, both forward to the

vacuole and backward to the Golgi apparatus (30–32).

406 Pigment Cell Res. 15, 2002

The third stage involves recycling, or transport from thePVC to the Golgi. This process requires the vps52p ⁄vps53p ⁄ vps54p complex (33), and the �retromer� complexconsisting of two subcomplexes: vps26p ⁄ vps29p ⁄ vps35p andvps5p ⁄ vps17p (34). All three complexes are conserved in

higher eukaryotes, suggesting an important role in endosome–Golgi retrieval.

The final stage in the CPY pathway consists of membrane

transport from the PVC to the vacuole. Proteins functioningin this process are the �Class B� and �Class C� vps proteins.�Class B� vps mutants (e.g. vps3p, vps5p, vps26p) showfragmented vacuoles, so this class of proteins probably

functions in late vacuole formation (30). �Class C� vpsproteins (vps11p, vps16p, vps18p and vps33p) interact witheach other and are localized at the vacuolar membrane.

�Class C� vps mutants also show fragmented vacuoles, so theproteins may be part of a complex functioning in the final

step of membrane transport to the vacuole (35). The �Class C�vps complex might also operate at other stages of thevacuolar transport pathway (36).

The ALP pathwayAlkaline phosphatase, a membrane-bound hydrolase, serves

as a prototypic protein for the ALP pathway (Fig. 1A).Alkaline phosphatase transport to the vacuole does notinvolve a prevacuolar compartment (37), and persists unal-tered in cells that are blocked in specific parts of the CPY

pathway. In fact, ALP leaves the Golgi complex via vesicleshaving a different membrane composition than that of CPY-containing vesicles (37, 38). Adaptor complex-3 (AP-3) is

Fig. 1. Simplified model of lysosomal biogenesis. (A) Yeast vacuolar and Drosophila pigment granule biogenesis pathways. Proteins known to regulatespecific steps in yeast transport are listed in white boxes. The list is not complete. The CPY pathway (upper arrows) transports vacuolar proteins in yeastfrom the Golgi through the PVC or MVB toward the vacuole. The ALP pathway (lower) carries vacuolar proteins directly to the vacuole, independent ofthe PVC. Transport of proteins toward the Drosophila pigment granule is blocked in several mutants, indicated in green boxes. (B) Mammalian lysosomeand lysosome-related organelle biogenesis pathways. Mouse mutants (pink boxes) and human mutants (yellow boxes) are indicated at their predicted siteof action in specific transport steps.


involved in the ALP pathway, although its precise site ofaction remains unclear (37, 39, 40). The syntaxin-likeSNARE, vam3p, is known to follow the ALP pathway to

the vacuole (37, 41). The vps41 and vps39p mutants areblocked in the processing of ALP, have nearly normal CPYdelivery, and accumulate structures that resemble mamma-lian MVB (38, 42).

The CPY and ALP pathways converge at the stepinvolving fusion with the vacuole. The two pathways sharegene products affecting vacuole targeting and fusion. In

addition, vam3p, the t-SNARE of the vacuole, functions inboth protein transport pathways to the vacuole (30, 41).Along with other proteins, vamp3p interacts with �Class C�vps gene products (35). Two other proteins whose mutationshave effects on both the ALP and CPY pathways are thedynamin homolog vps1 (43) and the rab7 homolog ypt7 (44).

Drosophila melanogaster

By the time evolution produced flies, specialized pigment cellshad developed to assist in vision. These cells, typified bythose in the eye of D. melanogaster, contain membrane-

bound organelles called pigment granules. Each pigmentgranule is filled with either ommochrome (brown) pigment,or drosopterin (red) pigment; the latter gives the Drosophila

eye its red color (45). More than 80 mutations affecting eyecolor in D. melanogaster have been described (45, 46). Someof the eye color genes encode pigment-synthesis enzymes(necessary for ommochrome or drosopterin production) or

ATP-binding cassette transporter (ABC) membrane trans-porters required for transport of pigment precursors. Incontrast, the �granule group� of mutant genes encodes

proteins that function in the delivery of proteins to pigmentgranules (45–47). These mutants have a reduction in bothtypes of pigment, as well as additional phenotypes unrelated

to eye color. Some of the granule group genes are homol-ogous to genes whose protein products are required forlysosomal biogenesis in other species. This became apparentwhen the d subunit of AP-3 was found to cause the garnet

phenotype (48, 49). Mammalian and yeast AP-3 complexeswere already recognized to mediate protein transport tolysosomal organelles (39, 50, 51). Later, deficiencies of the l,

b and r subunits of AP-3 were found to correspond to thegranule group mutants carmine, orange and ruby, respectively(52, 53), further supporting the concept that Drosophila

pigment granules, like yeast vacuoles, are related tolysosomes.

Gene defects have been established in three other members

of the granule group. The vps41, vps33 and vps18 genes aremutated in light, carnation and deep orange, respectively(54–56). The vps18p and vps33p genes are part of a multi-subunit complex that also contains vps11p and vps16p (35). A

mutation in any of these subunits in yeast results in accumu-lation of prevacuolar multivesicular bodies (35), similar to theaccumulation of multivesicular bodies found in pigment cells

of the Drosophila mutant deep orange (56) (Fig. 1A).The vps41p mutants in yeast also accumulate structures

that resemble mammalian MVB (38). This could be because

vps41p indirectly interacts with, and may function in thesame pathway with, the vps11p ⁄ vps16p ⁄ vps18p ⁄ vps33p

complex. Vps41p interacts with vps39p (57), vps39 yeastmutants show multivesiculated prevacuolar structures (42),and vps39p can bind to the vps11 subunit of the

vps11p ⁄ vps16p ⁄ vps18p ⁄ vps33p complex (57). In addition,in yeast and in humans, vps41p interacts with the d subunitof AP-3 and is required for formation of AP-3 coated carriervesicles (58, 59).

Identification of the gene defects in the granule groupmutants claret, lightoid, pink, and purploid may reveal othercritical components of the pigment granule ⁄ lysosome bio-

genesis pathway.

Mus Musculus

If Drosophila brought pigmented granules to the evolution-ary chain, mice contributed a group of specialized intracel-

lular vesicles. In general, the responsible genes have nohomologs in more primitive species, and their mutationsresult in phenotypes resembling human HPS. In fact, 14

genetically distinct mice display a combination of defects inmelanosomes (hypopigmenation), platelet dense bodies (pro-longed bleeding times), and in most cases, lysosomes (urinary

excretion of lysosomal enzymes) (60). The defective organ-elles are of lysosomal lineage, and the defects are comparablewith vacuolar transport defects in yeast and pigment granule

defects in Drosophila. Each mouse model exhibits autosomalrecessive inheritance, and the severity of the organelle defectsvaries among mutants (Table 1). The specific phenotypicfeatures of each mouse model may help predict the site of

action of the encoding protein as well as interactions amongproteins (Table 1 and Fig. 1B).

Pale ear ⁄ light earThe gene responsible for the pale ear mouse (ep) was foundby homology to the first human HPS gene, itself identified by

positional cloning (61–63). The HPS1 gene product has noknown function and has no homology to any other knownprotein. The pale ear mouse has a phenotype identical to thatof another mutant, light ear (le), whose gene was recently

identified as HPS4 (64). The HPS4 gene product also has noknown function and no homology to any known protein.Both ep and le mutants have similar coat colors, both show a

unique hypopigmentation of ears, tail and feet (60, 65), andboth manifest fewer and enlarged melanin granules indifferent cell types (62, 66, 67). In addition, le ⁄ le; ep ⁄ epdoubly homozygous mice have a phenotype identical to thatof the two singly homozygous mutants (68); this suggests thatthe proteins function in the same pathway.

No direct interaction between the HPS1 and HPS4 geneproducts has been shown, but studies in transfected melan-oma cells demonstrated partial colocalization in vesicularstructures in the perinuclear area (64). In addition, tissues of

the le mouse show no HPS1 protein expression (64), againsuggesting that the HPS1 and HPS4 proteins function in thesame pathway and could possibly interact. In human

melanotic cells, the HPS1 protein resides in uncoated vesiclesand early stage melanosomes. It exists in two distinct high-molecular weight complexes (a �200 kDa cytosolic and a

>500 kDa membrane-bound complex) distributed amonguncoated vesicles, early stage melanosomes and the cytosol


(69). Identification of the other components of thesecomplexes will reveal more about the function and site of

action of the HPS1 protein, as well as its possible interactionwith the HPS4 protein.

Pallid ⁄muted ⁄ reduced pigmentThe pallid (pa) mouse shows hypopigmentation, prolongedbleeding time, elevation of kidney lysosomal enzymes,

deficiency of serum a1-antitrypsin activity, and abnormalotolith formation (60, 70, 71). Long-term survival is reducedcompared with that of wild-type mice, due mainly to lunglesions consisting of air space enlargement and destruction of

alveolar septa (70, 72). The pa mouse has reduced or absentmelanosomes, and the remaining melanosomes exist in earlydevelopmental stages (types I and II melanosomes), as

assessed by 3,4-dihydroxyphenylalanine (DOPA) reactivity(71, 73). In choroid cells, melanosomes appear aggregated inlarge membrane-bound granules (73).

The pa mouse is defective in the pallidin (Pldn) gene (74),which encodes pallidin, a small coiled-coil protein with nohomologs in mouse, Drosophila or yeast. Yeast two-hybrid

experiments showed interaction of pallidin with syntaxin 13(Stx13), a t-SNARE protein that mediates vesicle fusion anddocking (74). Stx13 interacts with early endosome antigen 1(EEA 1), an effector of Rab5 GTPase that helps tether early

endosomes prior to fusion (75). The STX13 ⁄ Pldn complexmay function as a premelanosomal t-SNARE assisting fusionof specific vesicles that traffic from the trans-Golgi to the

premelanosome, carrying the melanosomal proteins tyrosin-ase, Tyrp 1 and Tyrp 2 (76, 77).

Recent reports indicate that pallidin is a component of an

�200 kDa asymmetric protein complex named BLOC-1 forbiogenesis of lysosome-related organelles complex-1 (78).Although the molecular mass of BLOC-1 resembles that ofan HPS1 protein complex (69), coimmunoprecipitation

experiments failed to detect an association between pallidinand HPS1, suggesting that the HPS1 complex is distinct from

BLOC-1 (78). The lack of pallidin in muted (mu) and reducedpigment (rp) fibroblasts suggests that the mu and rp gene

products might encode components of BLOC-1 that arerequired for pallidin stability (78).

The muted gene, mutated in the muted mouse, codes for a

small coiled-coil protein (like pallidin), which has no homo-logs in yeast or Drosophila. Muted is distributed in avesicular pattern throughout the cell body and dendrites

(79). The number and size of melanosomes in the mutedmouse is decreased and the remaining melanosomes oftencontain unusual inclusions and lamellar bodies (79). The mumouse has balance defects caused by the absence of inner ear

otoliths (60, 79). The muted protein is present in drasticallyreduced concentrations in pallid and rp mouse fibroblasts(78), and coimmunoprecipitation experiments show an

interaction between pallidin and the muted protein, againsuggesting that the muted protein is a part of the BLOC-1complex (78).

The rp gene has not yet been identified. The rp mouse has aphenotype similar to that of pa and mu in its coathypopigmentation and prolonged bleeding time. rp, along

with the gunmetal mouse (see below), displays cutaneousrather than oculocutaneous albinism; eye pigment formationis normal (60).

Biogenesis of lysosome-related organelles complex-1 func-

tion appears independent of AP-3 activity, and no physicalinteraction between BLOC-1 and AP-3 is demonstrable bycoimmunoprecipitation. The phenotype of the mu ⁄ pe double

mutant is significantly more severe than that of each singlemutant, and pa and mu fibroblasts do not have enhancedplasma membrane trafficking of LAMP proteins, as shown

for mocha. A clue to the function of BLOC-1 might comefrom the interaction of pallidin with syntaxin-13, whichwould indicate a role in membrane fusion. Pallidin canassociate with actin filaments (78), which are abundant not

only in the plasma membrane area, but also in the region ofthe Golgi complex (80, 81).

Table 1. Genes associated with pigment dilution due to abnormal vesicle formation or trafficking

Human Mouse Drosophila Yeast

Gene name Gene product Mutantb Gene product Mutant Gene product Mutant Gene product Mutant pathwaya

HPS1 HPS1 HPS-1 MEP-1 (ep) pale ear CG12855 – – –ADTB3A AP3B1 HPS-2 Ap3b1 pearl CG11427 (rb) ruby APL6 ALPHPS3 HPS3 HPS-3 HPS3 cocoa CG14562 – – –HPS4 HPS4 HPS-4 HPS4 light ear CG4866 – – –Pallidin PLDN – Pldn pallid – – – –Muted MUTED – muted muted – – – –RGGTA RABGGTA – Rabggta gunmetal CG12007 – – –d-3 AP3D1 (d) – Ap3d1 (d) mocha CG11197 (d) garnet APL5 ALPl-3 AP3M1(l3A) – Ap3m1(l3 A) – CG3035 (l3) carmine APM3 ALPr-3 AP3S1 (r3A) – Ap3s1 (r3A) – CG3029 (r3) orange APS3 ALPVPS33 VPS33A – VPS33 – CG12230 (car) carnation VPS33 CPYVPS41 VPS41 – VPS41 – CG18028(lt) light VPS41 ALPVPS18 VPS18 – VPS18 – CG3093 (dor) deep orange VPS18 CPYRAB27A RAB27A GS Rab27A ashen GM10914 – (Ypt)? ?Myosin 5A MYO5A ES MYO5A dilute Myosin V – Myo2? ?Melanophillin MLPH – Mlph leaden – – – –LYST LYST CHS LYST beige dAKAP550

dCG9011––

BPH1 ?

a CPY, defect in carboxypeptidase Y pathway; ALP, defect in alkaline phosphatase pathway; ?, unknown.b GS, Griscelli syndrome; ES, Elejalde syndrome; CHS, Chediak–Higashi syndrome.


Pearl ⁄mochaTwo mouse coat color mutants, mocha (mh) and pearl (pe),

were found to carry mutations encoding the d and b3Asubunits of AP-3, respectively (82, 83), further emphasizingthe connection of AP-3 to the biogenesis of lysosome-related

organelles. Both mh and pe mice exhibit coat and eye colordilution (because of fewer and smaller melanosomes inpigment forming cells), prolonged bleeding times, and

decreased secretion of lysosomal enzymes (60). The mochamice also exhibit balance problems due to otolith defectswhich eventually lead to deafness (84), as well as neurologicdefects such as hyperactivity and seizures (82, 85).

Unlike mocha, pearl does not show neurologic abnormal-ities, and pearl has normal hearing and balance. This couldbe explained by normal expression in pearl of brain-specific

b3B. This isoform of b3A, also known as b-NAP, allows fornormal AP-3 function in the pearl brain (82, 86). The pearlmutant is unique among the mouse coat color mutants in

that it displays night blindness, i.e. a reduced visual sensi-tivity to dim light, possibly because of the lower density ofmelanosomes in the retina (60, 87).

Ruby eye, ruby eye-2, cocoaThe mouse models ruby eye (ru), ruby eye-2 (ru-2) and cocoa(coa) have the same distinct coat color, different from all

other mouse models (60). This might mean that these threegenes function as a complex or in the same vesicularprocessing step. While the ru and ru-2 genes await isolation,

the coa mouse gene has been identified as HPS3 (88).The ru gene has reduced numbers of melanocytes in the

retina, skin, and other melanized tissues, and the melanosomes

are spheroidal rather than ovoid (60); this suggests that the rugene product functions in a relatively late step of vesicleformation such as MVB formation (Fig. 1B) [MBVs are

intermediates in the endosomal pathway; they have a limitingmembrane inside of which small membrane vesicles reside(77)]. Mast cells of the ruby eye mouse exhibit a threefold

increase in the number and duration of transient fusion events.This implicates a function for ru in regulating closure of the cellfusion pores which connect the lumen of a secretory vesiclewith the extracellular environment during exocytosis (89).

The coamouse has a mild phenotype with normal lysosomalenzyme secretion by platelets and kidney cells. The coa genehas a high density of small, abnormally shaped, unmelanized

early melanosomes and few mature melanosomes, withdisorganized and granular matrices. The coa eye has a paucityof melanosomes in both the retinal pigment epithelium and the

choroid, which also contains excessive multilamellar bodiesand aberrant melanosomes. These features, together withlocalization of HPS3 throughout the cytoplasm (88), suggest a

function late in melanosome biogenesis.

GunmetalThe gunmetal (gm) mouse is unusual among the HPS models

in that it exhibits only cutaneous albinism, not oculocutane-ous albinism (90). The gm mouse also exhibits thrombocy-topenia, with approximately one-third the normal number of

platelets (91), and abnormal a granules which containstriated inclusions and decreased contents of fibrinogen,platelet factor 4, and von Willebrand factor. Excess levels of

von Willebrand factor are found in plasma of the gm mouse,suggesting a defect in trafficking ⁄ processing rather thendefective synthesis (90). The gm platelets are increased in size,and dense granule contents are decreased to approximately

50% of normal; the bleeding time is still significantlyincreased (Table 2) (60, 91). These features make gm resem-ble a ⁄ d storage pool deficiency (92) rather than classical

HPS. The gm gene, RGGTA, codes for rab geranylgeranyl

Table 2. Murine mutants with pigment dilution and storage pool deficiency

Mouse model

Mousechromosome ⁄position

Genedefect

Bleedingtimea

Lysosomaldysfunctionb Coat color

Otolithformationdefect

Melanosome(number andshape) Comments

Cappuccino (cno) 5 ⁄ ? Unknown >15 min + Unique – Fewer, mostlyearly stage

Cocoa (coa) 3 ⁄ 12.5 cM HPS3 >15 min – Similar to ru,ru-2

– Small, abnormalshape

Human HPS-3disease

Gunmetal (gm) 14 ⁄ 20.7 cM RABGGTA >15 min – Unique – ? No eye hypopigmentationLight ear (le) 5 ⁄ 60.0 cM HPS4 >15 min + Similar to ep – Mostly early stage,

normal shapeHuman HPS-4 disease

Mocha (mh) 10 ⁄ 43.0 cM AP3D1 >15 min + Unique + Fewer and smaller,normal shape

Imbalance, hyperactivity

Muted (mu) 13 ⁄ 21.0 cM muted >15 min + Unique + Fewer, irregular shape ImbalancePale ear (ep) 19 ⁄ 42.0 cM HPS1 >15 min + Similar to le – Mostly early stage,

normal shapeHuman HPS-1 disease

Pallid ( pa) 2 ⁄ 67.6 cM pallidin >15 min + Unique + Fewer, early stage,normal shape

Pearl ( pe) 13 ⁄ 47.0 cM ADTB3A >15 min + Unique – Fewer and smaller,normal shape

Human HPS-2 disease

Reduced pigment (rp) 7 ⁄ 2.0 cM Unknown >15 min + Unique – ? No eye hypopigmentationRuby eye (ru) 19 ⁄ 44.0 cM Unknown >15 min + Similar to ru-2 – Fewer, spheroidal shapeRuby eye-2 (ru2) 7 ⁄ 25.0 cM Unknown >15 min + Similar to ru,

coa– ?

Sandy (sdy) 13 ⁄ 23.0 cM Unknown >15 min + Unique – ?Subtle gray (sut) 3 ⁄ 16.4 cM Unknown 7.5 min – Unique – ?

a Normal bleeding time in mouse (C57BL ⁄ 6J strain) is 2.1 min (60).b Excretion of lysosomal enzymes (60).?, unreported.


transferase a, an enzyme that attaches geranylgeranyl groupsto small GTPases (i.e. rabs) (93) for insertion into mem-branes (94). The rab proteins play key roles in membrane

remodeling and trafficking (95).

Subtle gray, sandy, cappuccinoThe subtle gray (sut) mouse has an intermediate bleeding time

and mild pigment dilution. In sut, platelet dense granuleserotonin is reduced, but adenine nucleotide concentrationsare normal (60, 96). Lysosomal enzyme secretion is also

normal. The sut gene product may act in specialized vesicletrafficking rather than in vesicle formation, as lysosomalfunction is normal (Table 1) (60), and most platelet contents

are present. Mild coat hypopigmentation also points to amelanosomal transport defect. The sut locus, althoughsituated on mouse chromosome 3 syntenic to human

chromosome 3q24 (where HPS3 resides) is not the murinecounterpart of human HPS3 (97).

The sandy (sa) mouse has diluted pigment in its eyes andfur, and has a prolonged bleeding time. Platelet serotonin

levels are very low and platelet aggregation is impaired (98).Platelet dense granules are reduced in number and lysosomalenzyme secretion is decreased (98). The severity of the

phenotype indicates a role for the sa gene product early inmelanosome biogenesis.

The cappuccino (cno) mouse has severe coat hypopigmen-

tation, which resembles that of pallid, a prolonged bleedingtime, and decreased lysosomal enzyme secretion (60, 99). Thecno melanosomes are immature and decreased in number inboth the eyes and skin. Behavioral abnormalities, such as

head tilting and poor balance, suggest otolith defects in theinner ear (99). The cno gene has not been isolated, but it hasbeen shown that the cno gene product functions in a pathway

independent of AP-3 (99).

Homo sapiens; the Hermansky–Pudlak syndromes

While the mouse displays 14 types of hypopigmentation andplatelet storage pool deficiency, man has so far found only

four genes in himself that cause a similar constellation offindings (Table 2). More are likely to be discovered, of course.The distribution of patients among the four known subtypes

of HPS is skewed by the large number (�450) of north-westPuerto Rican patients homozygous for a founder mutation inHPS1. These individuals constitute approximately 70% of the

world’s known cases of HPS. If they are disregarded, thefrequency of each HPS subtype can be estimated from theNIH experience involving 52 patients outside of north-west

Puerto Rico. In this group, 14 (27%) have HPS-1, three (6%)have HPS-2, 15 (29%) have HPS-3, seven (13%) have HPS-4,and 13 (25%) have no known mutation.

We now describe the four known HPS subtypes on bothclinical and cell biologic bases.

Clinical findings

Clinically, oculocutaneous albinism and platelet storage pooldeficiency characterize all types of HPS, but some clinicalfindings are specific for certain subtypes. For example, HPS-1

and HPS-4 patients can suffer granulomatous colitis andpulmonary fibrosis, while HPS-3 patients can have colitis buthave no manifested pulmonary fibrosis to date (Table 3).

HPS-2 patients are too few and too young to reveal if theywill ever develop these complications (100, 101). Among andwithin HPS subtypes, the extent of hypopigmentation

(Fig. 2), visual acuity deficit and bleeding diathesis varieswidely, although HPS-3 appears milder with respect to thesesigns and symptoms (102). The dermatologic, ophthalmo-logic, and pulmonary findings of HPS-1 have been exten-

sively described (10, 11, 16); in fact, because of the relativelyhigh prevalence of HPS-1, the clinical characteristics of thissubtype have, in the past, defined HPS as a medical entity

(103).

Cell biologyHPS-1

The HPS1 protein shows no homology to any other knownproteins and its function is unknown. HPS1 exists as part of

two distinct large complexes in melanotic cells, i.e. an�200 kDa cytosolic and a >500 kDa membrane-boundcomplex, distributed among uncoated vesicles, early stage

melanosomes and the cytoplasm (69). Patients with HPS-1,who have no expression of the HPS1 gene on Northern blot(61), have melanocytes which are hypopigmented, withseverely decreased melanin content (0–50% of normal). In

intact HPS-1 melanocytes, tyrosine hydroxylase activity wasalmost half that of normal, but in cell lysates of HPS-1melanocytes it was within the normal range (104), indicating

a sorting defect rather than a synthesis defect.Morphologic investigations of HPS-1 melanosomes

employed staining with DOPA, which localizes functional

tyrosinase. These studies showed numerous premelanosomeslacking DOPA reaction product distributed throughout thecell body and dendrites (104), indicating that tyrosinase

sorting requires the HPS1 gene product. In addition,large membranous complexes were detected containingmembrane-bound chambers, unpigmented and pigmented

Table 3. Clinical findings in human HPS subtypesa

SubtypeOculocutaneousalbinism

Visualacuitydeficitb

Bleedingdisorder

Infectiousdiathesis

Pulmonaryfibrosis Colitis

HPS-1 +++ +++ +++ – +++ ++HPS-2 ++ ++ + + ? ?HPS-3 + ++ + – – ⁄ + ++HPS-4 +++ +++ +++ – +++ ++

a ), absent; +, mild; ++, moderate; +++, severe; ?, unknown (only three patients). Severity can vary considerably within each subtype.b Varies from 20 of 50 to 20 of 400.


melanosomes, irregular deposits of DOPA reaction product,and granular ⁄ amorphous material (Fig. 3). DOPA-positive

�rings�, delineated on either side by limiting membranes, werealso detected (104). In normal melanocytes, the intracellulardistribution of tyrosinase-related protein-1 (Tyrp1) and

granulophysin appeared in a fine granular pattern through-out the cell. In HPS-1 cells, in contrast, these proteinsappeared in large granules throughout the cytoplasm, pro-

bably representing the membranous structures and ⁄ or ring-like structures (104), again indicating a missorting of theseadditional melanosomal proteins. Missorting of Tyrp1 andtyrosinase was also demonstrated by transfecting normal

melanocytes with anti-sense HPS1 cDNA (105). All thesefindings suggest a function of HPS1 (likely as part of acomplex) in sorting and early vesicle (premelanosome)

formation from the trans-Golgi network.

HPS-2

The ADTB3A gene, defective in HPS-2 (106), encodes theb3A subunit of AP-3. Adaptor complexes (AP) are hetero-

tetrameric and are involved in vesicle ⁄membrane formationand trafficking. Adaptor complexes play a role in formationof coated vesicles, as well as in the selection of cargo for these

vesicles. Adaptor complex-3, contains d, l, and r subunits inaddition to b3A. The b3A subunit is thought to bind clathrin,

whose rigid triskelion structure causes outpouching from anexisting membrane. In this fashion, AP-3 mediates theformation of vesicles such as the melanosome and platelet

dense body.Clues to the specific role of AP-3 in vesicle formation come

from studies of fibroblasts and melanocytes deficient in b3A.

In HPS-2 fibroblasts, deficient in AP-3 activity, trafficking oflysosomal proteins (e.g. LAMP-1, LAMP-2, and LAMP-3)through the plasma membrane is enhanced, suggesting thatthe plasma membrane provides a default pathway which

operates when normal AP-3 function is blocked (106). InHPS-2 melanocytes, tyrosinase expression is reduced andlimited to multivesicular bodies in the perinuclear region

(Fig. 4B). However, expression of Tyrp1 in HPS-2 melano-cytes is unaffected (107). These results suggest that traffickingof tyrosinase but not Tyrp1 is mediated via AP-3. Transfec-

tion of HPS-2 melanocytes with ADTB3A cDNA restoredb3A activity and cured the melanocytes of their tyrosinasemislocalization, confirming that AP-3 functions to traffic

tyrosinase to melanosomes (107). This helps explain thehypopigmentation of HPS-2 patients.

Fig. 2. Skin and hair hypopigmentation in patients with HPS-1 (A), HPS-2 (B), HPS-3 (C), and HPS-4 (D).


HPS-3

The function of the HPS3 protein is unknown, and HPS3has no homology to any known protein. However, HPS3

has a putative clathrin binding domain and potentialdileucine sorting signals, whose importance to HPS3function remains to be determined (108). HPS3 melanocytes

do not show obvious ultrastructural abnormalities. How-ever, they do contain some melanosomes with relatively

minimal DOPA reaction product as well as numerous50 nm DOPA-positive vesicles throughout the dendrites(Fig. 5). This is in agreement with the relatively mild

phenotype of HPS-3 patients (102) and the cocoa mouse.

Fig. 4. HPS-2 melanocytes are characterized by late endosome ⁄ multivesicular body-like structures. Melanocytes cultured from (A) a control individualor (B and C) patients with HPS-2 were processed for DOPA histochemistry. (A) Multivesicular bodies with minimal (1) or no (2) reaction product wereoccasionally present in control melanocytes. (B) In contrast, multivesicular bodies with much reaction product (arrows) were abundant in HPS-2melanocytes. (C) The HPS-2 multivesiculated bodies exhibited finger-like protrusions of their limiting membranes (1) and the DOPA positive reactionproduct appeared as aggregates (2), and ⁄ or within vesicles (3). (scale bars: A and B ¼ 0.4 lm; C ¼ 0.25 lm) [images adapted from Huizing et al. (107)].

Fig. 3. HPS-1 melanocytes are characterized by membranous complexes. Melanocytes cultured from patients with HPS-1 were processed with (A and C)or without (B) DOPA histochemistry to localize functional tyrosinase. (A) Perinuclear (cell at bottom) and dendritic (cell at top) area of two adjacentmelanocytes demonstrating 50-nm DOPA-positive vesicles (arrowheads) around the trans-Golgi network of the perinuclear area (cell at bottom),throughout the cytoplasm of the dendritic area (cell at top) and in the vicinity of a DOPA-positive membranous structure (arrowhead with asterisk).Some melanosomes contained (arrows) or were devoid of (arrows with asterisks) DOPA reaction product (N ¼ nucleus; open arrow ¼ membranouscomplexes; scale bar ¼ 1.0 lm). (B) High magnification of a membranous complex in the dendritic area of a melanocyte not treated with DOPAhistochemistry demonstrating limiting membranes (arrowheads) incompletely surrounding the complex and demarking an apparent cisternal space(asterisk). Within the complex are irregular profiles of limiting membranes (arrows) and two putative early stage melanosomes (stars) (scalebar ¼ 0.35 lm). (C) High magnification of a membranous complex in a melanocyte treated with DOPA histochemistry demonstrating two-unitmembranes (arrowheads) delineating a cisterna that can be filled with (arrows) or devoid of (arrow with asterisk) DOPA reaction product, indicating thepresence or absence of tyrosinase, respectively. At some sites, the opposing unit membranes appear to coalesce and extrude DOPA reaction product frombetween them (arrowheads with asterisks) or curve out of the plane of sectioning (brackets). Melanosomes in the vicinity or within the membranousprofiles can be filled with (open arrows) or devoid of (open arrows with asterisks) DOPA reaction product. Neighboring 50-nm diameter vesicles can alsocontain (arrows with black stars) or be devoid of (arrows with white stars) DOPA reaction product (scale bars ¼ 0.2 lm) [images adapted from Boissyet al. (104)].


HPS3 most likely functions in a late step of lysosome ⁄mel-anosome biogenesis.

HPS-4

There is evidence that the recently identified HPS4 protein,which has no known function and no homology to otherproteins, interacts with HPS1 (64). To date, no ultrastruc-

tural studies have been performed on HPS-4 melanocytes.Preliminary immunofluorescence data show accumulation ofLAMP-1 and LAMP-3 in large aggregates in the perinuclear

area in HPS-4 fibroblasts, similar to findings for HPS-1fibroblasts (M. Huizing, unpublished data). HPS4 might bepart of a high molecular weight complex that includes HPS1.

Related disorders

Several rare genetic disorders of hypopigmentation resemble

HPS. Choroideremia patients, with defects in a rab-escortprotein, share visual defects with HPS patients (109), andpatients with the Gray Platelet syndrome (gene defect

unknown) show abnormalities in a granules rather thandense granules (110). Chediak–Higashi syndrome (CHS) ischaracterized by variable hypopigmentation of skin, hair,

and eyes, a bleeding diathesis, progressive neurologic dys-function, and severe immunologic deficiency (111–113). TheCHS cells show giant lysosomes and lysosome-like organelles(114). The CHS melanocytes also contain giant, morpholog-

ically normal melanosomes with reduced pigmentation andaberrant trafficking and secretion of tyrosinase (115). Thegene defective in CHS is LYST, which encodes a large

protein (�430 kDa) with unknown function localized to thecytoplasm (116). How LYST defects change lysosomemorphology ⁄ shape is unknown, but alterations in several

biochemical pathways (e.g. protein kinase C) have beenimplicated (113). The beige mouse is the murine counterpartof human CHS (116).

Griscelli syndrome (GS) shows hypopigmentation of

the skin, silvery gray hair, immune abnormalities, and

hemophagocytic syndrome (117, 118). Another disorder,Elejalde syndrome (ES), is characterized by a similar pheno-

type, but has neurologic complications and no immunologicdefects (119). Griscelli syndrome is caused by mutations inRAB27A, a small guanosine triphosphate-binding protein

involved in targeting and fusion of transport vesicles (118,120). The mouse ashen is the murine counterpart of GS (121).Elejalde syndrome (with the murine counterpart dilute) is

caused by mutations in MYO5A, an actin-binding motorprotein (120, 122). The three mouse models, leaden [causedby mutations in melanophilin (123)], ashen, and dilute form aunique group with hypopigmented coat color caused by

pigment clumping in hair shafts (60). Their gene products,rab27A, myosin 5A, and melanophilin, have been shown tofunction in melanosome transport down dendrites for cap-

ture within the dendritic tips (124).

Molecular biology of HPS

Mutations in all four known human HPS causing genes havebeen identified (8). Most patients have mutations in HPS1,which has an open reading frame of 2103 bp, is divided

into 20 exons, and is located on chromosome 10q23.1–23.3(61). The first and most common mutation in HPS1 is a16-bp duplication in exon 15, responsible for HPS in north-west Puerto Rico (61). Thirteen other mutations have

been reported (8) and we recently found four new mutations(125).

HPS-2 disease is caused by mutations in ADTB3A (101,

106), which has an open reading frame of 3281 bp, is dividedinto 27 exons, and has been mapped to chromosome 5q13.2(101). Four mutations in three patients have been reported

(101, 106).HPS-3 disease is caused by mutations in HPS3 (108),

which consists of 17 exons, has a 3015-bp open readingframe, and has been mapped to chromosome 3q24 (108). A

founder mutation (3904-bp deletion) has been discovered incentral Puerto Rico (108) and a different founder mutation

Fig. 5. HPS-3 melanocytes are characterized by extensive distribution of 50-nm diameter, DOPA-positive vesicles. Melanocytes cultured from a patientwith HPS-3 were processed for DOPA histochemistry. (A) Golgi area (G) of the cell body demonstrating reaction product in the trans-Golgi network(white arrowheads) and 50-nm vesicles (black arrowheads). Reaction product was absent (arrows with asterisks) or present (arrows) from melanosomesin the vicinity. (N ¼ nucleus; scale bar ¼ 2.5 lm). (B) Dendritic area demonstrating numerous DOPA-positive melanosomes, few relatively DOPA-negative melanosomes (arrows with asterisks), and an abundance of 50-nm diameter, DOPA-positive vesicles (arrowheads) scattered throughout thecytoplasm (N ¼ nucleus; scale bar ¼ 2.5 lm).


(IVS5 + 1G fi A) in Ashkenazi Jewish HPS patients. Fiveother HPS3 mutations have been reported (102).

The HPS4 gene was recently found to cause HPS (64).

HPS4 has an open reading frame of 2127 bp, consists of 14exons, and has been mapped to chromosome 22q11.2–q12.2.Five different mutations have been reported (64).

A significant subset of HPS patients do not have mutations

in HPS1, ADTB3A, HPS3 or HPS4.

Protein: complexes and interactions

The progressive sophistication of specialized intracellular

vesicles, as evolution proceeds from yeast to man, reflects aseries of vertical relationships between the genes of onespecies and the next (Table 2). In addition, horizontalrelationships exist among the proteins responsible for form-

ing vesicles out of extant membranes. In particular, variousprotein complexes are known to play roles in proteintrafficking and vesicle formation. Examples already men-

tioned include the interactions of the HPS1 and HPS4proteins, the BLOC-1 complex, and the AP-3 complex.Others, noted below, deserve further investigation.

AP-1, AP-2, AP-4Although AP-3 mutations cause one type of HPS (HPS-2),

three other APs are known to exist. The AP-1 complex isubiquitously expressed in mammals, and mutations appear tobe lethal (126). Adaptor complex 1 binds clathrin and acts invesicle formation at the trans-Golgi. Adaptor complex-2

mediates endocytosis from the plasma membrane and isubiquitously expressed in mammals. Its function in lyso-somal biogenesis appears to be minimal. Adaptor complex-4

(127, 128) is not expressed in yeast or Drosophila, so itsfunction may be rather advanced. It is associated with thetrans-Golgi network (127, 128), but appears to function on

non-clathrin coated vesicles (126–128).

VPS complexesThe complex of the four �Class C� vps genes, vps11p ⁄vps16p ⁄ vps18p ⁄ vps33p, is conserved from yeast to man.Mutations in one of several subunits cause lysosomalbiogenesis defects in yeast and Drosophila. We hypothesized

that these four genes would be candidates to cause HPS inhumans, and we identified the human homologs of thesubunits (129). However, we found no mutations in any of

these genes in our HPS patients. The vps39p and vps41pmutants also form a complex in yeast (57) and are conservedin evolution. However, we also failed to find mutations in

vps41 and vps39 in HPS patients.

Other complexes

Many other known and unknown protein complexes playimportant roles in specific vesicular transport steps in

lysosomal biogenesis. Several recent reviews address thefunction and interactions of the Rab ⁄YPT family (120),SNAREs (131), syntaxins (132), the cytoskeleton (actin and

microtubuli) (133), and membrane lipid composition (134).Understanding these and other processes will help reveal themechanism of lysosomal biogenesis.

DISCUSSION

Evolution from yeast to higher eukaryotes has introduceddifferent lysosome-related organelles into specialized cells.Examples include melanosomes in melanocytes, dense gran-

ules in platelets, lytic granules in T lymphocytes and naturalkiller cells, basophilic granules in basophils and mast cells,azurophilic granules in neutrophils, and class II major

histocompatibility complex glycoproteins (MIICs) inantigen-presenting cells such as macrophages, dendriticepithelial cells and B lymphoblasts. These dedicated vesiclesin specialized cells are not necessary in yeast, but are essential

for more developed organisms. Hence, genes required foroverall cell viability are conserved from yeast throughhumans, while genes producing specialized organelles of the

lysosomal pathway, e.g. �HPS genes�, are present only inhigher eukaryotes.

How did nature deal with the need to acquire particular

genes for the maintenance of specialized organelles? First,some single copy yeast genes could have multiple isoforms ineukaryotes to achieve diversification of function. Examples

are yeast vps33, which has mammalian vps33a and vps33bisoforms (129) and the tissue specific isoforms b3A and b3Bof AP-3 (86). Secondly, some yeast orthologues could changetheir domain organization in higher eukaryotes as a result of

adaptation during evolution. For example, human vps41 hastwo alternative splice forms; one is membrane-associated andcontains a C-terminal RING-H2 sequence motif, while the

other splice form lacks this motif and is primarily cytoplas-mic (59). Thirdly, lysosome biogenesis in higher eukaryotescould rely upon novel genes having no homologs in yeast.

This appears to be the case for the HPS1, HPS3, HPS4,pallidin, and muted genes.

Of course, lysosome-related organelles, such as the mel-anosome and platelet dense granule, coexist with conven-

tional lysosomes in the same cell (77). Furthermore, theintracellular vesicles we regard as lysosomes really consist ofmany distinct functional groups. One of these is the secretory

lysosome, which shares several characteristics with melano-somes and platelet dense granules (135). All three have asingle limiting membrane, share common integral membrane

proteins by virtue of a common Golgi origin, and undergosecretion. All these attributes, which differentiate them fromother intracellular compartments, require sorting mecha-

nisms to ensure proper targeting of components to eachorganelle.

The genes that achieve this specialized protein andmembrane sorting and targeting are, in general, HPS genes.

HPS1, HPS3, and HPS4 are not evolutionarily conservedand have no homologs in yeast, although ADTB3A, respon-sible for HPS-2, does. Even in this case, however, the

specialized nature of vesicle formation is evident. The yeastAP-3 complex mediates transport of alkaline phosphatase tothe vacuole but does not contribute to the trafficking of

vps10p, the receptor of the vacuolar CPY. Similarly, mam-malian AP-3 mediates lysosomal targeting of LAMP-1,LAMP-2, and LIMP-2 or CD63, but is not involved in

MPR transport. In the melanocyte, it appears that AP-3mediates tyrosinase targeting to the melanosome, but is notinvolved in Tyrp1 transport (107).


Some clues to the stage at which HPS genes operate arisefrom the phenotypes of HPS model mice (60). In theseanimals, abnormal urinary secretion has been found for

lysosomal but not for non-lysosomal enzymes. This points toa late, post-Golgi (i.e. post-early sorting vesicle formation)stage of granule biogenesis or secretion, rather than to earlierstages at which lysosomal and non-lysosomal enzymes are

not yet sorted. In addition, Swank et al. (84) tested protonpump activity, necessary to maintain an acidic pH inlysosome-like organelles. No abnormalities in acidification

were noted in HPS mouse mutants, absolving this process asa cause of vesicular dysfunction. We have studied HPS-1,HPS-3, and HPS-4 fibroblasts with the fluorescent probe

LysoTracker Red (Molecular Probes, Eugene, OR, USA),which quantifies acidity, and also found no defects inlysosomal pH (M. Huizing, unpublished data).

Although protein deficiencies are likely responsible formost aberrations in vesicular trafficking, some attentionshould be paid to defects of phospholipid membranecomposition (134). Fusion pores, critical for membrane

fusion events, are closed by virtue of slight changes in thelipid composition of the pore (136), and post-Golgi sortingevents are likely mediated by phosphoinositides (137). Hence,

altered phospholipid levels may result in improper vesicleformation or trafficking.

Future investigations into HPS-causing genes will draw

from basic genetic and cell biologic principles. For example,mice can be bred to be doubly homozygous for two HPS-causing genes. If the double mutant shows a phenotypesimilar to that of the homozygous single mutants, it is likely

that their gene products interact and ⁄ or act in the samepathway. If the double mutant phenotype is more severe thanthat of the single homozygous mutants, then the gene

products likely function in separate pathways. In addition,examining the morphology of an HPS patient’s melanocytesat the electron microscope level may indicate the location of

the protein defect. Some illustrative findings might includeMVB formation, fragmented vacuoles, or accumulation ofsmall vesicles.

It took millions of years for yeast to rise to the level ofman, and for pigment vesicles to be bred into specializedcells. Yet the secrets of intracellular organelle formation arelikely to be revealed within a single human lifetime. The

process has begun.

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Hermansky-Pudlak Syndrome: Vesicle Formation from Yeast to Man

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