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Secretory Pathway

Karen J. Colley

University of Illinois at Chicago, Chicago, Illinois, USA

The eukaryotic cell is separated into several functionally

distinct, membrane-enclosed compartments (Figure 1). Each

compartment contains proteins required to accomplish speciﬁc

functions. Consequently, each protein must be sorted to its

proper location to ensure cell viability. Proteins possess speciﬁc

signals, either encoded in their amino acid sequences or added

as posttranslational modiﬁcations, which target them for the

various compartments of the cell. The pioneering work of

Dr. George Palade provided scientists with their ﬁrst picture of

the functional organization of the mammalian secretory path-

way. Later work showed that the secretory pathway acts as a

folding, modiﬁcation, and quality control system for proteins

that function in the endoplasmic reticulum (ER) and Golgi

apparatus, and for those that are targeted to the lysosome,

plasma membrane, and extracellular space. This article will

focus on protein targeting to and within the compartments of

the secretory pathway, and how proteins within this pathway

function to ensure that correctly folded and modiﬁed proteins

are delivered to the cell surface and secreted from cells.

Targeting of New Proteins to

the Secretory Pathway

WHAT KINDS OF PROTEINS

ARE

TARGETEDTOTHE

SECRETORY PATHWAY?

The proteins that are targeted to the secretory pathway

can be separated into two groups – those that function

in the ER and Golgi to ensure proper protein folding and

modiﬁcation (i.e., resident proteins), and those that are

processed in the ER and Golgi, and are transported to

later compartments like the lysosome, plasma mem-

brane, and extracellular space (Figure 1). Each of these

proteins not only possesses a signal to enter the secretory

pathway, but also may have a secondary signal to

localize it to a particular organelle within the pathway.

SIGNALS AND MECHANISMS OF

SECRETORY PATHWAY ENTRY

The 1999 Nobel Prize in physiology or medicine was

awarded to Dr. Gu

nter Blobel for his contributions to

our understanding of the mechanism of secretory path-

way entry. Dr. Blobel and his colleagues found that in

order to enter the secretory pathway, proteins are

synthesized with an amino terminal signal peptide that

allows them to cross the membrane of the endoplasmic

reticulum (ER). The signal peptide is recognized by a

complex of proteins and ribonucleic acid called the

signal recognition particle (SRP) (Figure 2). As the signal

peptide emerges from the ribosome during translation,

SRP binds to it and halts translation, and then targets the

new protein–ribosome complex to the cytoplasmic face

of the ER membrane where it binds to the SRP receptor.

Subsequently, the new protein–ribosome complex is

released from SRP and its receptor, and transferred to an

aqueous membrane channel known as the “translocon.”

Translation resumes and the new protein is co-transla-

tionally transferred through the translocon into the

lumen of the ER, where in many cases the signal peptide

is cleaved by a speciﬁc signal peptidase (Figure 2).

SOLUBLE AND INTEGRAL

MEMBRANE PROTEINS

Soluble proteins are completely translocated across the

ER membrane into the lumen (Figure 2). These proteins

will either remain in the ER, be targeted to another

organelle, or be secreted from the cell. Integral

membrane proteins that possess one or more hydro-

phobic membrane-spanning regions will use these

sequences to insert into the membrane of the ER and

either stay as ER-resident transmembrane proteins, or be

targeted to another cellular membrane. A type-I

membrane protein has a cleavable signal peptide and a

separate hydrophobic stretch of amino acids that acts as

a membrane-spanning region. This type of protein has

its amino terminus in the lumen of an organelle or the

outside of the cell (which are topologically equivalent),

and its carboxy terminus in the cytoplasm (Figure 2). In

contrast, a type-II membrane protein has an uncleavable

signal peptide, or signal anchor that is not at the

protein’s amino terminus and serves a dual function as

both signal peptide and a membrane-spanning region.

Type-II membrane proteins employ a more elaborate

insertion mechanism than do type-I membrane proteins.

For this reason, a type-II membrane protein will have its

carboxy terminus in the lumen of an organelle or outside

the cell, and its amino terminus in the cytoplasm

(Figure 2). Other proteins span the membrane several

times and are called type-III membrane proteins. They

can start with either cleavable signal peptides or

uncleavable signal anchors and possess variable num-

bers of hydrophobic membrane-spanning segments.

Protein Folding and Modiﬁcation

in the ER

THE INITIATION OF PROTEIN N-LINKED

GLYCOSYLATION IN THE ER

As proteins enter the ER lumen, they fold and assemble

with the help of chaperone proteins. Many proteins are

also co-translationally modiﬁed by the addition of

carbohydrates to asparagine residues in the process of

N-linked glycosylation (Figure 2). A preformed oligosac-

charide, consisting of three glucoses, nine mannoses, and

two N-acetylglucosamine residues (Glc

Man

GlcNAc

)

is transferred to accessible asparagine residues in the

tripeptide sequence asparagine-X-serine or threonine

(X cannot be proline) by the oligosaccharide protein

transferase complex. Subsequent modiﬁcation by glyco-

sidases (enzymes that remove monosaccharides) and

glycosyltransferases (enzymes that add monosacchar-

ides) in the ER and Golgi lead to the remodeling of the

N-linked oligosaccharides. These N-linked carbo-

hydrates help proteins fold, protect them from proteo-

lytic degradation and, in some cases, are critical for

modulating and mediating protein and cell interactions

at the cell surface and in the extracellular space.

CHAPERONES AND THE ER QUALITY

CONTROL SYSTEM

An important function of the ER is to serve as a site of

protein folding and quality control. Protein folding in

the ER includes the formation of intra-molecular

disulﬁde bonds, prolyl isomerization, and the sequestra-

tion of hydrophobic amino acids into the interior of the

protein. Protein disulﬁde bonds are formed as the

protein exits the translocon and may at ﬁrst form

incorrectly between cysteine residues close together

in the protein’s linear amino acid sequence. Thiol-

oxidoreductases, such as protein disulﬁde isomerase

(PDI), help to form and reorganize proteins’ disulﬁde

bonds into the most energetically favorable conﬁgur-

ation. Different types of chaperones monitor a protein’s

FIGURE 1 Compartments of eukaryotic cells and the organization of the secretory pathway. Diagrammatic representation of the compartments

of the eukaryotic cell is shown. The anterograde ﬂow of membrane and protein trafﬁc in the secretory pathway is shown in the box. Anterograde

ﬂow is indicated by arrows. Retrograde ﬂow between the ER and Golgi, endosome/lysosome system and Golgi, and plasma membrane and Golgi

does occur, but is not shown.

12 SECRETORY PATHWAY

folding and prevent exit of unfolded and unassembled

proteins from the ER. The chaperone BiP, originally

identiﬁed as an immunoglobulin heavy-chain-binding

protein, interacts with the exposed hydrophobic

sequences of folding intermediates of many proteins

and prevents their aggregation. Two chaperones called

calnexin and calreticulin recognize a monoglucosylated

carbohydrate structure (Glc

Man

GlcNAc

)thatis

formed by a special glucosyltransferase that recognizes

unfolded or misfolded proteins and adds a single glucose

to the Man

GlcNAc

structure. Proteins that are not

folded properly or are not assembled into oligomers with

partner subunits, are prevented from exiting the ER by

chaperone interactions, and can be targeted back across

the ER membrane through the translocon into the

cytoplasm where they are degraded by the proteosome

complex in a process called ER associated degradation

(ERAD).

Protein Transport through and

Localization in the Secretory

Pathway

VESICULAR TRANSPORT BETWEEN

THE

ER AND GOLGI

Proteins move between the ER and Golgi in vesicular

carriers. These vesicles are coated with speciﬁc sets of

cytoplasmic proteins that form the COP-I and COP-II

coats. COP-II-coated vesicles move from the ER to the

FIGURE 2 Entry into the secretory pathway. Many proteins are targeted for the secretory pathway by an amino terminal hydrophobic signal

peptide that allows their co-translational translocation across the ER membrane. [1] Signal recognition particle (SRP) recognizes the new protein’s

signal peptide. [2] The ribosome–new protein– SRP complex interacts with the SRP receptor on the cytoplasmic face of the ER membrane. [3] The

ribosome–new protein complex is transferred to the translocon channel, protein synthesis continues and the protein moves through the aqueous

channel. [4] As the new protein enters the lumen of the ER, its signal peptide is cleaved by the signal peptidase, chaperone proteins bind to aid in

folding and oligosaccharide protein transferase complex (OST) transfers oligosaccharides (arrowheads) to asparagine residues in the process of

N-linked glycosylation. [5] Soluble proteins lack additional hydrophobic sequences and are translocated through the translocon to complete their

folding and modiﬁcation in the ER lumen. [6] Type-I integral membrane proteins have a second hydrophobic sequence that partitions into the lipid

bilayer and acts as a membrane-spanning segment. These proteins have their amino termini in the lumen of an organelle or outstide the cell and their

carboxy-termini in the cell cytoplasm. [7] Unlike proteins with cleavable amino-terminal signal peptides, type-II integral membrane proteins have

an uncleavable signal anchor that target the protein to the secretory pathway and then partitions into the lipid bilayer to act as a membrane-

spanning segment. These proteins have their amino termini in the cell cytoplasm and their carboxy-termini in the lumen of an organelle or outside

the cell. Soluble and integral membrane proteins that enter at the level of the ER need not stay there, and can be transported out of the ER to other

locations in the pathway (see Figure 1, Secretory Pathway box).

SECRETORY PATHWAY 13

intermediate compartment (IC)/cis Golgi, while COP-I-

coated vesicles move from the Golgi back to the ER and

may also mediate transport between Golgi cisternae in

both the anterograde (toward the plasma membrane)

and retrograde (toward the ER) directions (Figure 3).

The process of vesicular transport can be separated into

three stages—cargo selection and budding, targeting,

and fusion. In the ﬁrst stage, the COP coats serve to

select cargo for exit from a compartment and help to

deform the membrane for vesicle budding. They

assemble on the membrane with the help of small

GTPases called ARF (speciﬁc for COP I) and Sar1p

(speciﬁc for COP II). After vesicle budding, the

hydrolysis of GTP by ARF and Sar1p leads to the

uncoating of the vesicle. This uncoating reveals other

vesicle proteins that are essential for vesicle targeting

and fusion. In the second and third stages, tethering

proteins on the transport vesicle and target membrane

interact weakly bringing the membranes together. This

allows vesicle-associated SNARE proteins and target

membrane-associated SNARE proteins to form com-

plexes. Subsequent conformational changes in the

SNARE protein complex bring the membranes together

for fusion. Another group of small GTPases (Rabs)

control the process of vesicular transport at several

levels by recruiting and activating various proteins in the

pathway.

PROTEIN LOCALIZATION IN THE ER

Proteins involved in protein folding, modiﬁcation, and

quality control must remain in the ER, while proteins

destined for the Golgi, lysosome, cell surface or those

that are secreted from the cell must exit. Exit from the

ER is a selective process that involves cargo receptors

that interact with COP-II coat components (Figure 3). It

is likely that most resident ER proteins are not selected

to exit the ER. It is clear, however, that some resident

proteins escape from the ER and are retrieved from the

Golgi and intermediate compartment by COP-I vesicles

(Figure 3). These proteins have speciﬁc amino acid

signals that allow their incorporation into COP-I

vesicles either by direct interaction with COP-I

components or indirectly by interaction with cargo

FIGURE 3 Comparison of two models of protein transport through the Golgi apparatus. In the vesicular transport model cargo proteins move

between the cisternae in vesicles, while Golgi enzymes are retained in their resident cisternae. [1] COP-II-coated vesicles transport new proteins

from the ER to the intermediate compartment (IC). [2] Resident ER proteins that escape the ER can be retrieved from the IC in COP-I-coated

vesicles. [3] COP-I-coated vesicles also transport anterograde cargo proteins between the Golgi cisternae in both a retrograde and anterograde

fashion (“percolating vesicles”). In the cisternal maturation model, cargo proteins enter a new cisterna at the cis face of the stack, and are modiﬁed

(matured) by “resident” Golgi enzymes that are continuously transported in a retrograde fashion into the sequentially maturing cisternae. [1a]

COP-II-coated vesicles transport new proteins from the ER to the IC where a new cis cisterna forms. [2a] Resident ER proteins that escape the ER

can be retrieved from the IC in COP-I-coated vesicles. [3a] Golgi enzymes are transported in a retrograde fashion in COP-I-coated vesicles to modify

the cargo proteins in earlier cisternae. Mechanisms of protein exit from the TGN are common to both models: [4] clathrin-coated vesicles mediate

late endosome (LE)-lysosome transport, while [5] other proteins are secreted in either a regulated or constitutive fashion to the plasma membrane or

extracellular space.

14 SECRETORY PATHWAY

receptors. For example, mammalian BiP is a soluble ER

protein that has the carboxy-terminal four amino acid

sequence lysine–aspartate–glutamate–leucine (KDEL).

This KDEL sequence is recognized by the KDEL

receptor that mediates their incorporation into COP I

vesicles moving from the intermediate compartment (IC)

back to the ER.

PROTEIN MODIFICATION IN THE GOLGI

The Golgi apparatus consists stacks of ﬂattened cister-

nae that contain enzymes and other proteins involved in

the further modiﬁcation and processing of newly made

proteins. It is separated into cis, medial, and trans

cisternae, followed by a meshwork of tubules and

vesicles called the trans Golgi network (TGN). The

process of N-linked glycosylation is completed through

the action of glycosidases and glycosyltransferases

localized in speciﬁc cisternae. Likewise, the glycosyla-

tion of serine and threonine residues (O-linked glycosy-

lation) is accomplished by other glycosyltransferases.

Additional modiﬁcations also occur in the Golgi. For

example, proteins and carbohydrate are sulfated by

sulfotransferases and some proteins are phosphorylated

on serine and threonine residues by Golgi kinases. In

addition, proteins like digestive enzymes (trypsin,

carboxypeptidase) and hormones (insulin) are made as

inactive precursors that must be proteolytically pro-

cessed to their active forms in the late Golgi or post-

Golgi compartments.

TRANSPORT OF PROTEINS

THROUGH THE

GOLGI

Currently there are two different models to explain

protein transport through the Golgi (Figure 3). The

vesicular transport model proposes that proteins move

sequentially between the Golgi cisterna in COP-I-coated

vesicles, while the cisternae themselves are stationary.

Proteins not retained in the cis Golgi, for example, would

be incorporated into coated vesicles and be transported to

the medial Golgi, and then to the trans Golgi. Proteins

destined for post-Golgi compartments move through

successive Golgi cisternae in this fashion, being modiﬁed

by the resident enzymes in each compartment (Figure 3).

In the cisternal maturation model a new cisterna is

formed on cis face of the Golgi stack from ER-derived

membrane. This requires both the anterograde transport

of newly synthesized proteins from the ER in COP-II-

coated vesicles and the retrograde transport of cis Golgi

enzymes from the pre-existing cis cisterna in COP I-

coated vesicles. The new cis cisterna and its contents

progressively mature through the stack as resident Golgi

enzymes are successively introduced by COP-I coated

vesicles (Figure 3). In the vesicular transport model, the

resident Golgi enzymes are retained in the cisternae while

the cargo moves in vesicles between the different

cisternae. In contrast, in the cisternal maturation

model, the “resident” enzymes are continuously moving

in a retrograde fashion, while the anterograde cargo

remains in the cisternae. Evidence for both mechanisms is

compelling, suggesting that both mechanisms may work

in parallel.

LOCALIZATION OF RESIDENT

GOLGI ENZYMES

In the context of the vesicular transport model, Golgi

enzymes are retained in speciﬁc cisternae. Two mechan-

isms have been suggested for Golgi protein retention.

The “bilayer thickness” mechanism suggests that the

relatively short transmembrane regions of Golgi proteins

prevent their incorporation into the wider, cholesterol-

rich lipid bilayers of the transport vesicles destined for

post-Golgi compartments (such as the plasma mem-

brane), and as a result, these proteins are retained in the

relatively cholesterol-poor Golgi. The “oligomeriza-

tion” mechanism predicts that once an enzyme has

reached its resident cisterna it forms homo- or hetero-

oligomers that prevent its incorporation into transport

vesicles moving to the next compartment. In the context

of the cisternal maturation model, resident Golgi

enzymes are actively incorporated into COP-I vesicles

for retrograde transport to a new cisterna, and one

might predict that the cytoplasmic tails of these proteins

would interact with COP-I-coat components to allow

vesicle incorporation. Interestingly, there are only a few

examples where the cytoplasmic tail of a Golgi enzyme

plays a primary role in its localization, whereas the

membrane-spanning regions of these proteins seem to be

more critical. Again, it is possible that some or all of

these mechanisms work together to maintain the steady-

state localization of the resident Golgi proteins.

Protein Exit from the Golgi and

Targeting to Post-Golgi Locations

PROTEIN EXIT FROM THE GOLGI

Once proteins reach the TGN they are sorted to post-

Golgi compartments that include the lysosome, the

plasma membrane, and the extracellular space

(Figure 3). Trafﬁcking to the lysosome–endosome system

involves clathrin-coated vesicles similar to those that

function in the uptake of proteins in endocytosis. In

contrast, transport to the plasma membrane, or exocy-

tosis, can occur either constitutively in secretory vesicles/

tubules or in a regulated fashion from secretory granules

found in speciﬁc cell types.

SECRETORY PATHWAY 15

PROTEIN TARGETING TO THE LYSOSOME

The lysosome is a degradative compartment that

contains numerous acid hydrolases that function to

digest proteins, lipids, and carbohydrates. The trafﬁck-

ing of the majority of lysosomal enzymes to the lysosome

requires mannose 6-phosphate residues on these

enzymes’ N-linked sugars. The mannose 6-phosphate

residues are recognized by receptors in the TGN that

mediate the incorporation of the new lysosomal enzymes

into clathrin-coated vesicles destined for the late

endosome compartment (Figure 3). These clathrin-

coated vesicles move from the TGN and fuse with the

late endosome, where a decrease in lumenal pH causes

the lysosomal enzymes to dissociate from the mannose

6-phosphate receptors. The enzymes are then trans-

ported to the lysosome, while the receptors recycle to the

TGN. Some lysosomal membrane proteins are also

trafﬁcked in clathrin-coated vesicles to the lysosome like

the soluble enzymes but without the use of a mannose

6-phosphate marker, while others are transported to the

cell surface, incorporated into a different set of clathrin-

coated vesicles used in the process of endocytosis, and

then trafﬁcked to the lysosome via the late endosome.

CONSTITUTIVE AND REGULATED

SECRETION

In many cell types, membrane-associated and soluble

proteins move to the plasma membrane constitutively

without a requirement for speciﬁc signals. Constitutively

secreted proteins include receptors, channel proteins,

cell adhesion molecules, and soluble extracellular matrix

and serum proteins. Other proteins like hormones and

neurotransmitters are targeted to secretory granules that

are involved in regulated secretion from endocrine and

exocrine cells, some types of immune cells, and neurons.

These granules remain in a secretion-ready state

until extracellular signals that lead to an increase in

intracellular calcium levels trigger the exocytosis of

their contents.

FURTHER READING

Ellgaard, L., Molinari, M., and Helenius, A. (1999). Setting the

standards: Quality control in the secretory pathway. Science 286,

1882–1888.

Farquhar, M. G., and Palade, G. E. 1998. The Golgi apparatus: 100

years of progress and controversy. Trends Cell Biol. 8, 2–10.

Intracellular compartments and protein sorting (chapter 12) and

intracellular vesicular trafﬁc (chapter 13). In The Molecular

Biology of the Cell (B. Alberts, A. Johnson, J. Lewis, M. Raff, K.

Roberts, and P. Walter, eds.), 4th edition, pp. 659–766. Garland

Science, New York.

Kornfeld, S., and Mellman, I. (1989). The biogenesis of lysosomes.

Annu. Rev. Cell Biol. 5, 483–525.

Palade, G. (1975). Intracellular aspects of the process of protein

synthesis. Science 189, 347– 358.

Rapoport, T. A., Jungnickel, B., and Kutay, U. (1996). Protein

transport across the eukaryotic endoplasmic reticulum and

bacterial inner membranes. Annu. Rev. Biochem. 65, 271– 303.

Rockefeller University web site describing Dr. Gu

nter Blobel’s Nobel

Prize research (http://www.rockefeller.edu).

BIOGRAPHY

Karen J. Colley is a Professor in the Department of Biochemistry and

Molecular Genetics at the University of Illinois College of Medicine

in Chicago. She holds a Ph.D. from Washington University in St.

Louis, and received her postdoctoral training at the University of

California, Los Angeles. Her principal research interests are in

protein trafﬁcking and glycosylation. Her recent studies focus on

the elucidation of the signals and mechanisms of Golgi glycosyl-

transferase localization.

16 SECRETORY PATHWAY

Selenoprotein Synthesis

August Bo

University of Munich, Munich, Germany

Selenoproteins contain one or more residues of the nonstan-

dard amino acid selenocysteine, which is an analogue of

cysteine in which a selenol group replaces a thiol. The majority

of these proteins catalyze some oxidation/reduction function

in which the selenol of the selenocysteine that is present in the

active site of the respective enzyme takes part in the reaction.

The advantage of having a selenol instead of a thiol lies in the

fact that it confers to these enzymes a higher kinetic efﬁciency.

In some biological systems, selenoproteins may also fulﬁll a

structural role because of their capacity to oligomerize proteins

via the formation of diselenide or mixed disulﬁde– selenide

bridges. The biosynthesis of selenoproteins is unique since the

incorporation of selenocysteine occurs co-translationally by

the ribosome and not posttranslationally. Selenocysteine

insertion is DNA encoded, requires the function of a cognate

tRNA and of a speciﬁc translation elongation factor different

from elongation factor Tu. Selenocysteine, therefore, has been

designated as the 21st amino acid.

Bacterial Selenoprotein Synthesis

The structure and the function of the components

involved in selenocysteine biosynthesis have been charac-

terized to a considerable extent in the case of the bacterial

system. The process can be divided into three functional

steps, namely the biosynthesis of selenocysteine in the

tRNA-bound state, the formation of a complex between

elongation factor SelB, GTP, selenocysteyl-tRNA

Sec

and

the mRNA, and the decoding event at the ribosome. As

far as it is known, though there are some major

differences, similarities also exist between bacterial

selenoprotein synthesis and the process characteristic of

eukarya and archaea.

SELENOCYSTEINE BIOSYNTHESIS

Figure 1 summarizes the process of selenocysteine

biosynthesis as it has been worked out for Escherichia

coli. It requires the activities of three enzymes, namely

seryl-tRNA synthetase (SerS), selenophosphate synthe-

tase (SelD), and selenocysteine synthase (SelA) plus the

speciﬁc tRNA (tRNA

Sec

). Seryl-tRNA synthetase

charges tRNA

Sec

with L-serine, selenocysteine synthase

converts the seryl-tRNA

Sec

into selenocysteyl-tRNA

Sec

using selenophosphate as a source for activated sel-

enium. Selenophosphate is provided by selenophosphate

synthetase from selenide in an ATP-dependent reaction.

The genes for these components had been identiﬁed with

the aid of E. coli mutants isolated by Mandrand–

Berthelot as being pleiotropically deﬁcient in formate

dehydrogenase activities.

tRNA

Sec

tRNA

Sec

(Figure 2) is the key molecule of selenoprotein

synthesis since it serves both as the adaptor for

selenocysteine biosynthesis and for incorporation of the

amino acid at the ribosome. It deviates in size, secondary

structure, and in normally conserved sequence positions

from canonical elongator tRNA species. Because of the

elongated extra arm and the one base-pair-extended

aminoacyl acceptor arm, tRNA

Sec

species are the largest

members of the elongator tRNA family. All tRNA

Sec

species identiﬁed thus far possess a UCA anticodon which

enables them to pair with UGA stop codons (but only if

these are in a special mRNA sequence context). More-

over, tRNA

Sec

species deviate from canonical elongator

tRNA species in sequence positions which are usually

invariant and which are involved in the establishment of

novel tertiary interactions within the molecule.

On the basis of its serine identity elements, tRNA

Sec

charged by the cellular seryl-tRNA synthetase which

also aminoacylates serine inserting isoacceptors. How-

ever, both the afﬁnity and the rate of aminacylation are

diminished in comparison to the charging of tRNA

Ser

resulting in an overall 100-fold reduced efﬁciency.

Selenocysteine Synthase

The overall reaction catalyzed by selenocysteine

synthase consists in the exchange of the hydroxylgroup

of the serine moiety of seryl-tRNA

Sec

by a selenol

group (Figure 1). The reaction occurs in two steps; ﬁrst,

the amino group of serine forms a Schiff base with

the carbonyl of the pyridoxal 5

-phosphate cofactor of

selenocysteine synthase leads to the 2,3-elimination of a

water molecule and the formation of dehydroalanyl-

tRNA

Sec

and second, nucleophilic addition of reduced

selenium to the double bond of dehydroalanyl-tRNA

Sec

from selenophosphate as a donor yields selenocysteyl-

tRNA

Sec

Selenocysteine synthase from E. coli is a homodeca-

meric enzyme and low resolution electron microscopy

revealed that it is made up of two pentameric rings

stacked on top of each other. Two subunits each are able

to bind one molecule of seryl-tRNA

Sec

, so the fully

loaded enzyme can complex ﬁve charged tRNA mol-

ecules. As serine isoacceptor tRNAs are not recognized,

the tRNA must have determinants for the speciﬁc

recognition of seryl-tRNA

Sec

by selenocysteine synthase

and antideterminants for the rejection of seryl-tRNA

Ser

species. The speciﬁcity for discrimination of the sel-

enium donor is not as strict since the puriﬁed enzyme

accepts thiophosphate instead of selenophosphate as a

substrate. This results in the formation of cysteyl-

tRNA

Sec

. So the discrimination between sulfur and

selenium must take place at some other step of

selenocysteine biosynthesis.

Selenophosphate Synthetase

Puriﬁed selenocysteine synthase does not exhibit an

absolute requirement for selenophosphate, as a substrate

to convert seryl-tRNA

Sec

into selenocysteyl-tRNA

Sec

since the reaction also occurs in the presence of high

concentrations of selenide. Even sulﬁde is accepted

although at a very low efﬁciency. So, the necessity for

selenophosphate as a substrate may reside in one or

more of the following three aspects, i.e., (1) to

discriminate sulﬁde from selenide, (2) to efﬁciently use

low concentrations of selenium compounds, and (3) to

accelerate the reaction rate effected by the activation of

the trace element. Indeed, selenophosphate synthetase

efﬁciently discriminates between sulﬁde and selenide,

and thus excludes sulfur from intrusion into the

selenium pathway. Selenophosphate synthetase from

E. coli is a monomeric enzyme with a unique reaction

mechanism since formally it transfers the

-phosphate of

ATP to selenide with the intermediate formation of

enzyme-bound ADP which is subsequently hydrolysed

into AMP and inorganic phosphate.

FORMATION OF THE SelB 3

GTP 3 S

ELENOCYSTEYL-TRNA 3

SECIS C

OMPLEX

Elongation factor Tu, which forms a complex with all 20

standard aminoacyl-tRNAs and donates them to the

ribosomal A-site, displays only minimal binding afﬁnity

FIGURE 1 Path of selenocysteine biosynthesis. For explanation see text.

18 SELENOPROTEIN SYNTHESIS

for selenocysteyl-tRNA

Sec

. Consequently, insertion of

selenocysteine requires the function of an alternate

elongation factor which is SelB. SelB from E. coli is a

70 kDa protein which contains the sequence elements of

elongation factor Tu in the N-terminal two-third of the

molecule (designated domains I, II, and III) plus a

domain IV of about 25 kDa which can be subdivided

into domains IVa and IVb. Domains I, II and III share

their functions with those of elongation factor Tu,

namely the binding of guanosine nucleotides and of

charged tRNA. An important difference, however, is that

they can discriminate between the serylated and the

selenocysteylated forms of tRNA

Sec

. In this way, the

insertion of serine instead of selenocysteine, which

would lead to an inactive enzyme, is prevented. The

structural basis for this discrimination ability has not yet

been resolved. A second difference is that the overall

afﬁnity for GTP is about 10-fold higher than that for

GDP which obviates the need for the function of a

guanosine nucleotide release factor since GDP is

chemically replaced by GTP. In accordance, the structure

of SelB lacks those subdomains which in elongation

factor Tu are responsible for interaction with the GDP

release factor EF-Ts. The 25 kDa C-terminal extension

of SelB (domain IV) is required for the function in

selenoprotein synthesis since its truncation inactivates

the molecule. The reason is that subdomain IVb binds to

a secondary structure of the mRNA (the SECIS element)

coding for selenoprotein synthesis. SelB, thus, is able to

form a quaternary complex with GTP and two RNA

ligands, namely selenocysteyl-tRNA and the SECIS

element (Figure 3). The isolated domains IV or IVb

retain the binding capacity for the SECIS element.

Formation of the quaternary complex follows random

order kinetics. An important feature also is that the

stability of the complex is increased when both RNA

ligands are bound.

The SECIS element itself is a hairpin structure formed

within a section of 39 bases of the selenoprotein mRNA

which follows the codon specifying selenocysteine

insertion at the 3

-side. Binding of SelB takes place to

its apical stem loop minihelix of 17 nucleotides. Genetic

and structural analysis showed that bases in the loop

region plus a bulged-out U in the helix are required for

the interaction with SelB. This apical part of the SECIS

element is separated by a short unpaired region from a

helix at the base of the hairpin. Pairing within this

second helix is not essential but it increases the efﬁciency

of selenocysteine insertion. An absolute requirement,

FIGURE 2 Cloverleaf presentation of the structure of tRNA

Sec

from

E. coli. Modiﬁed bases are shaded. Tertiary interactions via base

pairing are indicated by connecting red lines, and those involving

intercalation are denoted by arrows. Bases and base pairings deviating

from the consensus are indicated in green.

FIGURE 3 Translation of prokaryotic selenoprotein mRNA. Note that the SECIS element is within the mRNA reading frame and is complexed to

domain IVb of SelB carrying selenocysteyl-tRNA

Sec

and GTP.

SELENOPROTEIN SYNTHESIS 19

however, is that the codon determining selenocysteine

insertion lies within a critical distance relative to the

binding site of SelB.

Bacterial SECIS elements lie within the reading frame

of their selenoprotein mRNAs; they are thus subject to

stringent sequence constraints in order to deliver a

functional gene product. However, they do not need to

be translated since they also function when placed in the

-untranslated region at the correct distance to the

selenocysteine codon within an upstream reading frame.

The sequence constraint (which depends on the protein

to be formed) and the requirement for binding of SelB

restricts the number of selenoprotein mRNAs to be

expressed in a single organism and explains why the vast

majority of selenoprotein genes cannot be heterolo-

gously expressed unless the cognate SelB gene is

coexpressed. Thus, SelB and their SECIS elements are

subject to coevolution.

DECODING EVENT AT THE RIBOSOME

In all biological systems analyzed thus far, selenocysteine

insertion is directed by the opal stop codon UGA but

only if it is followed by an SECIS element at the correct

distance. This violates the dogma that no codon can

have more than one meaning within a single cell. The

questions to be answered therefore are: (1) what

prevents the UGA to be used as a termination signal,

and (2) which mechanism interferes with insertion of

selenocysteine at ordinary UGA stop codons?

Counteraction of Stop at the UGA?

A convincing answer to the question why the

selenocysteine-speciﬁc UGA codon does not function

as an efﬁcient termination signal must await structural

information on the decoding complex. It is, however,

clear that termination always competes with seleno-

cysteine insertion, especially under conditions when

the capacity for decoding the UGA with selenocysteine

is a limiting factor. This can be, for example, a surplus

of selenoprotein mRNA in relation to the amount of

SelB quaternary complex which forces the ribosome

to stall at the UGA. One fact identiﬁed to be involved

in the suppression of termination is that the base

following the UGA at the 3

-side in selenoprotein

mRNAs is prodominately an A or C, which renders the

UGA a weak termination signal. Also, the two amino

acids preceding selenocysteine in the nascent polypep-

tide chain are predominantly hydrophobic which

counteracts the dissociation of the nascent polypeptide

from the ribosome, when translation pauses at a

“hungry” codon present in the A site. Additional

mechanisms, however, must exist which contribute to

the suppression of termination.

Selenocysteine Speciﬁcity of UGA Codons

From the colinearity between the mRNA nucleotide

sequence and the amino acid sequence of the translation

product, it is clear that UGA determines the position

where selenocysteine is to be inserted during translation.

The speciﬁcity of the UGA, however, is determined by

the codon context, i.e., by the existence of a SECIS

element at the 3

side. The results of extensive

biochemical and biophysical analysis suggest the follow-

ing scenario for the decoding process: (1) SelB forms the

quaternary complex at the mRNA in which the two

RNA ligands display cooperativity in their interaction

with the protein; (2) in this quaternary complex SelB

attains a conformation compatible for interaction with

the ribosome which then results in stimulation of GTP

hydrolysis by SelB which in turn causes the release of the

charged tRNA in the vicinity of the ribosomal A-site; (3)

loss of the tRNA ligand causes the SelB protein to return

to a conformation with about tenfold lower afﬁnity for

the SECIS element. As a consequence, the mRNA is

released from the protein and freed for the translation of

codons downstream of the UGA. The consequence of the

complex cascade of reactions is that the efﬁciency of the

decoding of UGA with selenocysteine is lower than that

of any of the standard sense codons. It is also reﬂected by

a considerable pause taking place when the ribosome

encounters the quaternary complex at the mRNA. In the

absence of selenocysteyl-tRNA, binding of SelB alone to

the mRNA does not retard the rate of translation.

Archaeal and Eukaryal

Selenoprotein Synthesis

tRNA

Sec

species from archaea and eukarya share several

structural similarities with the bacterial counterparts but

they are more related to each other than either one is to

bacterial tRNA

Sec

. There is also considerable sequence

similarity between selenophosphate synthetases from all

three lines of descent rendering their annotation in

genome projects easy. On the other hand, homologues

for the bacterial selenocysteine synthase have not been

identiﬁed yet in any of the genomic sequences from

organisms known to synthesize selenoproteins.

Whereas UGA directs selenocysteine insertion also in

archaea and eukarya, a fundamental difference is that

the SECIS element is not positioned within the reading

frame but in the 3

-nontranslated region of the mRNA.

SECIS elements from organisms of the three lines of

descent are different by sequence and by secondary

structure. They may be positioned at different distances

from the actual termination codon and/or the seleno-

cysteine inserting UGA codon but a critical distance

must not be underpassed. It is thought that the selective

value for having the SECIS element in the nontranslated

SELENOPROTEIN SYNTHESIS