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1 Genetics of Yeast Mannoprotein Biosynthesis CLINTON E. BALLOU

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Department of Biochemistry, University of California, Berkeley, CA 94720

In this paper I intend to summarize what we know about yeast mannoprotein structure, what features of the biosynthetic pathway have been defined, how genetic techniques have been useful in gaining this information, and what is known about the genetic control of mannoprotein biosynthesis. Yeast Mannoprotein Structure Prior to 1965, the limited information available was due mainly to the methylation studies of Haworth et al. (1) which i n dicated that yeast mannan was a highly-branched polysaccharide with 1->2, 1->3 and 1->6 linkages, in no specific order. Dramatic developments have occurred during the last 10 years that have revolutionized our understanding of the structure of this very complex glycoprotein we now c a l l a mannoprotein. The f i r s t im portant development was the acetolysis procedure, f i r s t applied by Gorin and Perlin (2) and then extended in a series of papers in our laboratory (3-10), that allowed selective cleavage of the 1->6 linkages in the mannan component of the glycoprotein and f a c i l i t a t e d the isolation of oligosaccharide fragments whose struc tures could then be established by classical methods. From a comparison of the acetolysis oliogsaccharide patterns (Figure 1) from different mannoproteins (6), i t was clear that the mannan structures were species-specific (11). The second important step in elucidation of mannoprotein structure was the discovery of an enzyme with the a b i l i t y to r e move the mannan sidechains selectively so that the backbone structure could be studied (12). As a consequence, several yeast mannans, although not all, have been shown to have a linear α1->6linked backbone (13, 14, 15). As a corollary, it follows that the acetolysis oligosaccharides represent sidechain fragments of the mannan, and by their characterization we were able to arrive at schematic structures for the mannan chains (Figure 2). These two developments f a c i l i t a t e d a third; namely, the e l u cidation of the immunochemistry of the yeast mannoproteins. 0-8412-0555-8/80/47-126-001$05.00/0 ©

1980 A m e r i c a n C h e m i c a l Society

Sandford and Matsuda; Fungal Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

FUNGAL POLYSACCHARIDES

2

FRACTION FROM BIO-GEL P-2 COLUMN Science

Figure 1. Acetolysis oligosaccharide patterns from different yeast mannopro teins (11)- The products from the reaction, which was carried out according to Kef. 6, were separated on a Bio-Gel P-2 column (2 X 200 cm) by elution with O.J M acetic acid. M to M are mannose to mannopentaose, M P is mannotriose phosphate, M^P is mannotetraose phosphate, and MfiNAc is N-acetylglucosamine linked to mannotetraose. 5

3

Saccharomyces i t a l i eus

Kloeckera b r e v i s

6

6

Ρ+ αΜ αΜ

6

6

6

αΜ-> αΜ+ αΜ+ αΜ+ αΜ+

j>2

^2

φ2

αΜ

αΜ αΜ

αΜ αΜ

^2

φ2

αΜ αΜ αΜ

αΜ +

^2

αΜ αΜ 3

+3

αΜ αΜ αΜ Kluyveromyces l a c t i s

Saccharomyces c e r e v i s i a e 6

6

6

6

^2

^2

^2

^2

^2

αΜ

6

Ρ+ αΜ αΜ αΜ αΜ + f + f αΜ αΜ αΜ αΜ 2

αΜ

αΜ αΜ

2

6

6

αΜ^Μ+ αΜ+ αΜ+ αΜ+

6

αΜ^ αΜ^ αΜ^ αΜ^ αΜ^

^2

j.2

j.2

αΜ αΜ αΜ

2

2

aGNAc+ aM

αΜ αΜ

αΜ αΜ

Figure 2. Structures for the mannoproteins whose acetolysis patterns are shown in Figure 1. The structures illustrate the nature of the backbone and sidechains but they are not intended to indicate the order or number of sidechains.


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BALLOU

Yeast Mannoprotein

Biosynthesis

3

Rabbit a n t i s e r a r a i s e d against i n t r a v e n o u s l y i n j e c t e d whole yeast c e l l s g i v e strong p r e c i p i t i n r e a c t i o n s w i t h i s o l a t e d mannopro t e i n s ( l 6 ) and the homologous r e a c t i o n s can u s u a l l y be i n h i b i t e d by a c e t o l y s i s fragments from the mannan (ΓΓ, l8_, 19., 20) . Such s t u d i e s r e v e a l e d t h a t the immunodominant s t r u c t u r e s o f the yeast c e l l surface are the mannoprotein s i d e c h a i n s , and t h a t some s i d e chains are more immunogenic than others (Figure 3 ) . The d i s c o v e r y t h a t the mannan component o f the yeast c e l l was the major surface determinant produced important d i v i d e n d s , f o r i t suggested a procedure by which mutants w i t h a l t e r e d manno p r o t e i n s might be obtained. The procedure i n v o l v e d the s e l e c t i o n , from a mutagenized yeast c u l t u r e , o f v a r i a n t s t h a t no longer were a g g l u t i n a t e d by antiserum prepared against the parent c e l l s (21). Because the o n l y phenotypic expression of such mutants i s a change i n the carbohydrate component of the mannoproteins, char a c t e r i z a t i o n o f the mutants r e q u i r e d extensive s t r u c t u r a l analy s i s o f the c e l l w a l l s (22_). The screening f o r c e l l surface mutants, as c o n t r a s t e d w i t h s e l e c t i o n or enrichment, can be done with a n t i s e r a o f d i f f e r e n t s p e c i f i c i t i e s (23) or with the A l c i a n Blue dye b i n d i n g assay {2k) t h a t w i l l detect the presence or ab sence o f phosphate i n the mannan. A recent improved procedure, r e p o r t e d at t h i s meeting ( 2 5 ) , d e s c r i b e s the use o f a f l u o r e s c e n t l e c t i n f o r the s e l e c t i o n and enrichment o f yeast mannoprotein mutants by a f l u o r e s c e n c e - a c t i v a t e d c e l l s o r t e r . T h i s procedure should be of general use i f f l u o r e s c e n t a n t i b o d i e s can be em ployed i n p l a c e o f l e c t i n . The v a r i o u s mannoprotein mutants w i l l be d i s c u s s e d i n a l a t e r s e c t i o n o f t h i s r e p o r t , but one mutant must be considered now because i t has played an extremely important r o l e i n e l u c i d a t i n g the s t r u c t u r e o f the mannoproteins. T h i s i s the mnn2 mutant, which i s c h a r a c t e r i z e d by an a c e t o l y s i s p a t t e r n t h a t r e v e a l s mostly mannose and i n d i c a t e s t h a t the mannoprotein has a predominantly unbranched al->6-linked backbone s t r u c t u r e ( 2 l ) . Studies by Dr. Tasuku Nakajima, while a p o s t d o c t o r a l v i s i t o r i n my l a b o r a t o r y , demonstrated t h a t the small amount o f branching s t i l l detected i n the mannoprotein represented the presence o f a core s t r u c t u r e t h a t l i n k e d the main mannan chain t o the p r o t e i n (Figure k) {26). A v e r y important c o n c l u s i o n o f t h i s study i s t h a t yeast mannoproteins and mammalian and p l a n t g l y c o p r o t e i n s have i d e n t i c a l a s p a r a g i n e - l i n k e d core carbohydrate s t r u c t u r e s ( 2 7 ) , and t h a t t h i s core s t r u c t u r e i s modified i n yeast by the a d d i t i o n o f a polymannose outer c h a i n . A f i n a l p o i n t o f i n t e r e s t i s the f a c t t h a t the yeast core s t r u c t u r e , even i n a homogeneous g l y c o p r o t e i n , i s heterogeneous ( 2 8 ) . Yeast Mannoprotein B i o s y n t h e s i s A number o f e a r l y s t u d i e s on mannan b i o s y n t h e s i s (29_, 30) were done without a c l e a r i d e a o f the s t r u c t u r e o f the substance and, consequently, were d i f f i c u l t t o i n t e r p r e t . With the



6

6

6

-*αΜ+ αΜ+ αΜ+

6

6

->αΜ+ αΜ+ αΜ->

6

-*αΜ+ αΜ-* αΜ->

Figure 3. Immunodominant sidechains in yeast mannoproteins. The curved lines indicate the antibody binding sites inferred from hapten inhibition studies. Refer to Figure 2 for the distribution of these determinants in different yeast manno proteins.

6

6

6

6

6

[aI^ aNk aNk aM+ aM] +

2

aM

+

2

+2

+2Π

αΜ

+

2

Φ

2

αΜ

6

6

^3

2

+3

+

3

|

3 +2

αΜ αΜ +3

^3

αΜ αΜ

OUTER CHAIN

f

αΜ αΜ αΜ αΜ +2

+

αΜ αΜ αΜ ^3

6

f

aM aM aM ««- Ρ +2

6

^ a^ aM^ aI^ aI^ B^BGNAc^BGNAc-vAsn

αΜ CORE ?Ι4* 2

al^ ?I4^ 2

I

2

^ a ^ ? ^ aM* aNk aM+ ?M* a

3

2

Ser(Thr)

2

ALKALI-LABILE OLIGOSACCHARIDES Figure 4. Structures of the carbohydrate chains linked to protein in Saccharo myces cerevisiae X2180 mannoprotein. Four types of mannose units are linked to serine (Ser) and threonine (Thr), whereas a more complex polysaccharide chain is attached to asparagine (Asn). All anomeric linkages are a, except for the trisaccharide unit βΜαη-+ βΟΝΑο-+ βΟΝΑο-+ by which the polysaccharide is linked to asparagine. The subscript η in the outer chain is 3 to 5. 4

4


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BALLOU

Yeast Mannoprotein

Biosynthesis

5

advantage o f h i n d s i g h t , we can now p r e d i c t that the process should be d i v i d e d i n t o two p a r t s : ( l ) the synthesis o f the s e r i n e - and t h r e o n i n e - l i n k e d o l i g o s a c c h a r i d e s and (2) the s y n t h e s i s o f the a s p a r a g i n e - l i n k e d u n i t s (Figure 5). Each o f these can be v i s u a l i z e d as having three or more phases, namely, initiation, elonga tion and termination, w i t h the p o s s i b i l i t y o f a terminal modifica tion step f o r the a s p a r a g i n e - l i n k e d u n i t s . From s t u d i e s p r i m a r i l y i n the l a b o r a t o r y o f Widmar Tanner, at Regensberg, the i n i t i a t i o n step both f o r the o l i g o s a c c h a r i d e chains l i n k e d to hydroxy amino a c i d s (31) and the p o l y s a c c h a r i d e chains l i n k e d t o asparagine (32) are known to i n v o l v e l i p i d c a r r i e r s . D o l i c h y l mannosylphosphate i s the donor f o r a d d i t i o n o f the f i r s t mannose to s e r i n e and t h r e o n i n e , whereas the subsequent mannoses are d e r i v e d from guanosine diphosphate mannose. B i o s y n t h e s i s o f the p o l y s a c c h a r i d e u n i t s l i n k e d to asparagine i s much more complex; the core u n i t i s f i r s t c o n s t r u c t e d as a d o l i c h y l - l i n k e d d e r i v a t i v e , apparently sim i l a r to t h a t i n v o l v e d i n s y n t h e s i s o f mammalian g l y c o p r o t e i n s (33), t h i s i s t r a n s f e r r e d to asparagine i n the p r o t e i n , and the outer c h a i n o f the p o l y s a c c h a r i d e i s formed by s e q u e n t i a l a d d i t i o n o f mannose u n i t s from guanosine diphosphate mannose. M o d i f i c a t i o n s o f the outer chain are observed i n many mannoproteins, and and i n two i n s t a n c e s that have been s t u d i e d they i n c l u d e a d d i t i o n o f mannosylphosphate u n i t s (3M and N-acetylglucosamine (35.) t o the mannan s i d e c h a i n s . Now t h a t the general aspects o f mannoprotein s t r u c t u r e and b i o s y n t h e s i s are manifest, s i g n i f i c a n t new advances w i l l depend on the p u r i f i c a t i o n o f the v a r i o u s g l y c o s y l t r a n s f e r a s e s and a study o f t h e i r r e a c t i o n parameters w i t h exogenous acceptors (36). An important aim should be to d e l i n e a t e the steps i n the assembly o f the l i p i d - l i n k e d core u n i t , i n c l u d i n g any p r o c e s s i n g o f the molecule (37) t h a t may occur p r i o r t o i t s t r a n s f e r t o the p r o t e i n . Whether the al->6 l i n k a g e i n the core i s made by the same enzyme t h a t makes t h i s l i n k a g e i n the outer chain must be answered, and the t r a n s f e r a s e t h a t adds mannose in.al->3 l i n k a g e t o the backbone o f the core must be d i s t i n g u i s h e d from the al-*3-mannosyltransfer ase t h a t adds t h i s u n i t i n t e r m i n a l p o s i t i o n s elsewhere i n the mannoprotein (36). Genetic

A n a l y s i s o f Yeast Mannoproteins

In t h i s s e c t i o n , I consider the v a r i a n t s i n Saccharomyces mannan s t r u c t u r e t h a t occur n a t u r a l l y and those t h a t have been produced by d i r e c t e d mutation. From a comparison o f t h e i r aceto l y s i s p a t t e r n s , three i n t e r f e r t i l e wild-type Saccharomyces species have been d i s t i n g u i s h e d . These d i f f e r o n l y i n the lengths o f t h e i r longest s i d e chains t h a t , i n t u r n , r e f l e c t the a c t i v i t i e s o f two presumably d i f f e r e n t al-K3-mannosyltransferases (Figure 6 ) . D i p l o i d s o f s t r a i n s A and Β have the Β phenotype, whereas d i p l o i d s o f e i t h e r A or Β w i t h C have the C phenotype, which suggests t h a t


6


-GNAc- •GNAc-ljl-M-M-M

ASN-GNAc-GNAc-IJI-IJI-IJI-M )

PROTEIN SYNTHESIS

ί

M M M 1 1 M M

? M

M

M

M

IJI-M-IJI-M-[-M-M-- Μ - Μ ]

SER-M

Ijl M

Γ

çrp

ASN-

Ijl ijl M

MM M

M

ASN-GNAc-GNAc-ljl-ljl-ljl-H

χ

U M M

V

1 M

(

MM M

\

M M

SER-M

SIDE CHAIN MODIFICATION

Figure 5. Postulated glycosylation pathway in mannoprotein biosynthesis. The core unit attached to asparagine is derived from Dolichol-P -GNAc-Man , where y is about 15, whereas the first mannose attached to serine is derived from Dolichol-P-Man. In the above figure, χ is about 3 to 5. 2

-Μ ι aM

2

+2 Figure 6. Structures of the longest sidechains in three wild-type interfertile Saccharomyces species. These structures support the existence of a natural poly morphism within the species and they are explained by two independently seg regating al -> 3-mannosyltransf erases along with inactive forms of each locus.

aM

y

-M-

-M+2

+2

aM

aM + +

aM

+2

2

aM

aM

+

3

3

aM +

3

aM

A

Β


C

1.

BALLOU

Yeast Mannoprotein

7

Biosynthesis

two d i f f e r e n t and dominant al-K3-mannosyltranferases are i n v o l v e d i n determining these s t r u c t u r e s ( l l ) . Several mannan mutants o f S. cevevisiae have been obtained by treatment with e t h y l methane s u l f o n a t e (Figure 7 ) (3§_). The mnnl mutant has the phenotype o f s t r a i n A and does not complement i t , which i n d i c a t e s t h a t they i n v o l v e the same l o c u s . T h i s muta t i o n i s o f p a r t i c u l a r i n t e r e s t because o f i t s p l e i o t r o p i c nature; that i s , the a d d i t i o n o f mannose i s a f f e c t e d i n s e v e r a l s t r u c t u r a l l y d i f f e r e n t l o c a t i o n s . In c o n t r a s t , the mnn2 andrnnnS( 3 9 ) mutations seem t o a f f e c t o n l y the outer c h a i n , as do the mnn4 and mnn6 (3*0 mutations, since mannosylphosphate u n i t s occur o n l y i n the outer c h a i n . The mnnZ mutation i s s i m i l a r t o mnn5 although i t a l s o a f f e c t s the s e r i n e - l i n k e d u n i t s . No mutant with a defect i n forming the αΐ+β-backbone has been observed. S e v e r a l o f the mannan mutants have been p l a c e d on the genetic map (Figure 8 ) , and i t i s notable t h a t the f i r s t two mapped were centromere-linked ( 2 3 kO). Centromere l i n k a g e i s p o s t u l a t e d t o favor r e t e n t i o n o f a gene by minimizing recombination. 9

9

Genetic

Control o f Mannoprotein

Biosynthesis

Several l e v e l s o f c o n t r o l must be i n v o l v e d i n mannoprotein b i o s y n t h e s i s (Table I ) . Because these are g l y c o p r o t e i n s , t h e Table I Levels o f C o n t r o l i n Mannoprotein

Biosynthesis

1. P r o t e i n s y n t h e s i s : t r a n s c r i p t i o n & t r a n s l a t i o n . 2.

G l y c o s y l t r a n s f e r a s e a c t i v i t y and l o c a l i z a t i o n .

3.

Amino a c i d acceptor

h. Synthesis 5.

sequences.

and p r o c e s s i n g o f mannan core.

Translocation o f glycosylated protein.

6. A d d i t i o n o f outer chain t o mannan core. 7·

Termination and m o d i f i c a t i o n o f outer

chain.

f i r s t and most fundamental c o n t r o l i s at the l e v e l o f p r o t e i n biosynthesis. Several c e l l w a l l enzymes, such as i n v e r t a s e and a c i d phosphatase, a r e subject t o c a t a b o l i t e r e p r e s s i o n ( U l ) , and the s y n t h e s i s o f these mannoproteins i s presumably c o n t r o l l e d by r e g u l a t i o n o f t r a n s c r i p t i o n and t r a n s l a t i o n . Sexual a g g l u t i n i n s , g l y c o p r o t e i n s c o n t r o l l e d by the mating type l o c u s , are probably under s i m i l a r r e g u l a t i o n (k2). The second l e v e l o f c o n t r o l o f mannoprotein b i o s y n t h e s i s i n v o l v e s the g l y c o s y l t r a n s f e r a s e s t h a t c o n s t r u c t the precursors and add them t o the p r o t e i n . We have a l r e a d y seen t h a t the d i s t r i b u t i o n o f such enzyme a c t i v i t i e s i n the w i l d p o p u l a t i o n o f Saochamyoes species v a r i e s i n a f a s h i o n reminiscent o f the enzymes


8


r UÎ L_î !_î '_ÎM—1 M

M

M

—•M'-ÎMi-ÎMi-ÎM'-ÎMÎ-ÎGNAc'-ÎGNAc—Asn

M

i : t: r\:

- - - - -------ivH-:--i-:---fr,, m n n 2

mnn5

x

M

M

M

M^P

M

M

M

---ΐτ-ΐτ-^iî, M

mnnl

M

1

M

i: t:

M L M _ _ m_nn4j _m η η 6

M

M

- f M M i M

M

Outer Chain

M

Core

Ser

M —M-

(Thr)

M —M— M2

Base-Labile Oligosaccharides Figure 7. Representation of alterations in S. cerevisiae mannoproteins obtained by direct mutations. The dashed lines indicate the parts of the molecule that are eliminated by different mutations. The pleiotropic effect of the m n n l mutation is particularly striking.

mnn2 gall,7,10

pet9

lys2

met8

_L_

II

mnnl org 9 ura3

trp2

hisl

mnn4 trp3 XI

J

ural I

dbll L _

metlé ^

metl L _

Figure 8. Chromosome locations of genetic loci concerned with biosynthesis of the carbohydrate part of S. cerevisiae mannoprotein. The circles indicate the centromeres.


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BALLOU

Yeast Mannoprotein

Biosynthesis

9

i n v o l v e d i n formation of the blood group substances i n mammals. The wide d i s t r i b u t i o n on the yeast chromosomes o f the genes cont r o l l i n g the known mannosyltransferases (23) shows t h a t they do not occur i n any o p e r o n - l i k e arrangement. A t h i r d l e v e l o f c o n t r o l i s probably exerted by the amino a c i d sequences that determine g l y c o s y l a t i o n s i t e s i n the p r o t e i n . In mammalian g l y c o p r o t e i n s , a l l asparagine g l y c o s y l a t i o n s i t e s occur i n a sequence -Asn-X-Ser(Thr)-, although a l l such s i t e s are not g l y c o s y l a t e d jk3). Moreover, i n some instances the same p r o t e i n may be synthesized w i t h or without the carbohydrate component being added; f o r example, r i b o n u c l e a s e A and B. As y e t , no p u b l i s h e d study o f yeast mannoprotein b i o s y n t h e s i s has d e a l t w i t h t h i s matter, nor has the s t r u c t u r a l requirement f o r g l y c o s y l a t i o n at s e r i n e and threonine r e s i d u e s i n a p r o t e i n been i n v e s t i g a t e d . An i n t e r e s t i n g example o f the l a t t e r type o f mannoprotein i s the sexual a g g l u t i n i n from Hansenula wingei type 5 - c e l l s . T h i s substance contains 10% p r o t e i n t h a t i s 55% s e r i n e and 9% t h r e o n i n e , and at l e a s t 90% o f these hydroxy amino a c i d s are g l y c o s y l a t e d (hk). Thus, about two-thirds o f the amino a c i d r e s i d u e s i n the p r o t e i n c h a i n c a r r y attached o l i g o s a c c h a r i d e s . A f o u r t h l e v e l o f c o n t r o l i n mannoprotein b i o s y n t h e s i s concerns the s t r u c t u r e o f the core o l i g o s a c c h a r i d e t h a t i s l i n k e d t o asparagine. This u n i t has been shown to be heterogeneous whether i s o l a t e d from a bulk c e l l w a l l mannoprotein p r e p a r a t i o n jk5), from a pure enzyme such as e x t e r n a l i n v e r t a s e (h6_), o r even from the i n t r a c e l l u l a r mannoprotein carboxypeptidase Y ( 2 8 ) . It i s not known whether t h i s heterogeneity i s r e a l or i s an a r t i f a c t o f mixing o f the i s o l a t e d o l i g o s a c c h a r i d e s t h a t are i n d i v i d u a l l y homogeneous at a p a r t i c u l a r g l y c o s y l a t i o n s i t e i n the p r o t e i n . Some support f o r the l a t t e r p o s s i b i l i t y i s the f a c t t h a t carboxypeptidase Y, which contains four carbohydrate chains, g i v e s four o l i g o s a c c h a r i d e s i n approximately equimolar amounts when d i g e s t e d w i t h endo-3-N-acetylglucosaminidase. I t i s a l s o p o s s i b l e t h a t the yeast mannoprotein core i s made as a homogeneous substance, which then undergoes a p r o c e s s i n g r e a c t i o n to y i e l d v a r i o u s homol o g s i n a manner s i m i l a r to t h a t already documented f o r mammalian g l y c o p r o t e i n chains (37)· A f i f t h , and perhaps one o f the most important l e v e l s o f c o n t r o l , concerns the f a c t o r s t h a t determine whether a g l y c o p r o t e i n to which the core o l i g o s a c c h a r i d e s have been added i s r e t a i n e d i n s i d e the c e l l , as carboxypeptidase Y i s , or i s f u r t h e r g l y c o s y l a t e d to give a mannoprotein that i s secreted, as e x t e r n a l i n v e r t a s e i s (kj). Based on what has been l e a r n e d i n other systems, the primary s i g n a l that determines u l t i m a t e d e s t i n a t i o n probably w i l l be found i n the p r o t e i n component, and i t seems u n l i k e l y t h a t the carbohydrate w i l l p l a y a determining r o l e ( ^ 8 , k9). The c r i t i c a l step may w e l l be the type o f packaging that occurs at the time o f g l y c o s y l a t i o n , and t h a t the molecules dest i n e d f o r the periplasm are secreted i n t o v e s i c l e s of the G o l g i


10


system whereas those to be r e t a i n e d i n the c e l l are s e c r e t e d i n t o the v a c u o l a r system ( 50_). A s i x t h l e v e l o f c o n t r o l must determine which o f the core u n i t s added t o a mannoprotein are elongated and which are not. Thus, e x t e r n a l i n v e r t a s e has 9-10 carbohydrate chains per p r o t e i n subunit (51.), and over h a l f o f them appear t o c o n s i s t s o l e l y o f the core u n i t ( U 6 ) . A simple e x p l a n a t i o n c o u l d be that these u n i t s are sequestered i n s i d e when the i n v e r t a s e f o l d s i n t o i t s n a t i v e conformation, and o n l y those core u n i t s s t i l l exposed on the surface o f the p r o t e i n have the outer c h a i n added. As a seventh, and perhaps f i n a l , l e v e l o f c o n t r o l , I come t o those f a c t o r s t h a t may determine the extent o f e l a b o r a t i o n and m o d i f i c a t i o n o f the mannan outer c h a i n s . The a c e t o l y s i s p a t t e r n s suggest that each yeast makes mannoproteins with a c h a r a c t e r i s t i c degree o f branching (6_), and i t i s p o s s i b l e t h a t the side chains occur i n some l o o s e l y r e p e a t i n g order ( 1 0 ) . T h i s would not r e q u i r e o u t s i d e i n f o r m a t i o n , such as t h a t s u p p l i e d by a template, but c o u l d r e s u l t from the c h a r a c t e r i s t i c a f f i n i t i e s o f the d i f f e r e n t g l y c o s y l t r a n s f e r a s e s f o r the changing s t r u c t u r e at the growing end o f the c h a i n ( 3 6 ) . Thus, the al-*6-mannosyltransferase c o u l d have a h i g h a f f i n i t y f o r a c h a i n terminated by a branch o f a l - * 2 - l i n k e d mannose u n i t s , but a very low a f f i n i t y f o r a c h a i n terminated by two or more u n s u b s t i t u t e d a l - * 6 - l i n k e d u n i t s . If the al+2-mannosyltransferase that i n i t i a t e s branching had the r e v e r s e s p e c i f i c i t y , t h i s would assure that branching and backbone s y n t h e s i s c o u l d proceed w i t h some r e g u l a r i t y . Other f a c t o r s c o u l d r e g u l a t e the l e n g t h o f the mannan c h a i n s . Because guanosine diphosphate mannose i s the donor, i t s a v a i l a b i l i t y would be c r i t i c a l and i s p o s s i b l y c o n t r o l l e d by the energy charge o f the c e l l ( 5 2 ) . I t appears t h a t mannoprotein molecules are s y n t h e s i z e d and processed at a f a i r l y constant r a t e , and that the molecules made i n a c e l l w i t h l i m i t e d mannose donor would be s e c r e t e d with l e s s than the normal amount o f carbohydrate. A l t e r n a t i v e l y , i n the mnn2 mutant that makes mannan without s i d e chains, the molecules appear t o be made with a somewhat l o n g e r backbone, which c o u l d r e s u l t from the a v a i l a b i l i t y o f excess mannose donor that was not used to form the s i d e c h a i n s . Thus, the e l o n g a t i o n process may operate l i k e an assembly l i n e , except that the l i n e does not stop i f the c e l l i s unable to add a p a r t i c u l a r sugar, or the model may be changed i f the u s u a l p a r t s are not available. The c e l l u l a r l o c a l i z a t i o n o f the d i f f e r e n t g l y c o s y l t r a n s f e r ases w i l l probably t u r n out to be o f v e r y great importance i n t h i s r e s p e c t . These enzymes are membrane-bound and may be d i s t r i b u t e d i n a nonrandom f a s h i o n i n the endoplasmic r e t i c u l u m and the G o l g i apparatus, perhaps i n some r e l a t i o n to the order i n which the d i f f e r e n t processes are c a r r i e d out ( 5 3 ) . A type o f mutant t h i s p r e d i c t s i s one t h a t s e c r e t e s an a l t e r e d mannan even though i t possesses the a p p a r e n t l y normal g l y c o s y l t r a n s f e r a s e a c t i v i t y i n s i d e the c e l l . We have obtained such a mutant i n


1.

BALLOU

Yeast Mannoprotein

11

Biosynthesis

Kluyveromyces lactis t h a t secretes mannan l a c k i n g the t e r m i n a l a l + 2 - l i n k e d N-acetylglueosamine u n i t s even though c e l l e x t r a c t s show t h e wild-type t r a n s f e r a s e a c t i v i t y when assayed with an exogenous acceptor (35.). A l t e r n a t i v e explanations f o r t h i s observ a t i o n have been considered (Table I I ) , and i t s t i l l may develop Table I I P o s s i b l e Defects i n Kluyveromyees Mannoprotein mnn2-2 Mutant

lactis

1. Temperature s e n s i t i v e enzyme. 2.

Enzyme w i t h a l t e r e d Km.

3.

A l t e r e d donor a v a i l a b i l i t y .

k. A l t e r e d acceptor s t r u c t u r e . 5.

Over production

6. Under production

o f an i n h i b i t o r . o f an a c t i v a t o r .

T- A l t e r e d l o c a l i z a t i o n o f enzyme. t h a t a defect i n enzyme l o c a l i z a t i o n i s i n v o l v e d (5^). Such def e c t s have been noted i n other systems i n which processes dependent on membrane-bound enzymes are d i s r u p t e d because t h e enzyme f a i l s t o bind t o the membrane o r i s i n c o r r e c t l y i n s e r t e d ( 5 5 ) . In S. cerevisiae, a s i m i l a r mannan m o d i f i c a t i o n r e a c t i o n occurs t h a t i n v o l v e s the a d d i t i o n o f mannosylphosphate u n i t s t o the t r i s a c c h a r i d e sidechains ( 3 M . The f u n c t i o n o f these u n i t s i s unknown but they could o b v i o u s l y c o n t r o l the charge on the c e l l s u r f a c e , which may be important i n some environmental s i t u a t i o n s . We have obtained two types o f mutants t h a t r e g u l a t e t h i s process. They segregate independently, but both r e s u l t i n a mannan without phosphate. One mutation {mnn4) ( 2 2 ) i s dominant and probably a f f e c t s a l o c u s t h a t r e g u l a t e s synthesis o f the mannosylphosphate t r a n s f e r a s e (Table I I I ) ; the other i s r e c e s s i v e Table I I I P o s s i b l e Defects i n Saccharomyces Mannoprotein mnn4 Mutant

cerevisiae

1. Over production

o f an i n h i b i t o r .

2.

Over production

o f a phosphodiesterase.

3.

Formation o f an a l t e r e d subunit t o negative complementation.

h. Formation o f an a l t e r e d

leading

repressor.

(mnn6) (3U_) and could be at the s t r u c t u r a l gene l o c u s f o r t h i s enzyme. A t h i r d l o c u s that r e g u l a t e s the amount o f phosphate i n



12

the mannan has been r e p o r t e d (2h). I t a l s o i s dominant and maps on the same chromosome as mnn4 although the two segregate independently ( 2 3 ) . The c h a r a c t e r i z a t i o n o f these t h r e e g e n e t i c l o c i may h e l p t o e l u c i d a t e the mechanism by which t h i s step i n mannoprotein biosynthesis i s regulated. 9

Concluding Remarks T h i s b r i e f summary o f the s t r u c t u r e , b i o s y n t h e s i s and genetic c o n t r o l o f yeast mannoproteins should, i f anything, p o i n t out t h e complexity o f the problem w i t h which we are d e a l i n g . Our present knowledge o f the s t r u c t u r e o f Saccharomyces cerevisiae mannoprot e i n s makes p o s s i b l e a l o g i c a l study o f i t s b i o s y n t h e s i s . The mannan mutants have f a c i l i t a t e d t h i s study, but, more importantly, they have suggested probable mechanisms f o r the o v e r a l l r e g u l a t i o n o f i t s b i o s y n t h e s i s , t r a n s l o c a t i o n and s e c r e t i o n . I t i s obvious t h a t t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f a d d i t i o n a l mutants w i l l be u s e f u l i n f u r t h e r i n g t h i s work, and i t i s an approach that i s s t r o n g l y recommended. Acknowledgement s T h i s work was supported by N a t i o n a l Science Foundation Grant P C M 7 7 - 2 7 3 3 U and U.S. P u b l i c Health S e r v i c e Grant A I - 1 2 5 2 2 . Literature

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