The 'Palade pathway'

Pioneers in the field of eukaryotic protein targeting were George

Palade and co-workers who carried out studies on the secretion of

chymotrypsinogen from pancreatic cells of the guinea pig. Some o f this

early work is described in a review article by Palade (1975) and will be

summarised here. Initially, Palade and co-workers used electron microscopy to

study the pancreatic cells of the guinea pig. From histological studies in the

1950's, hypotheses were formed about the possible functioning o f the

pancreatic cells. They were observed to be 'packed with stacked endoplasmic

reticulum (ER) cisternae which were studded with ribosomes'. In 1959, using

cell fractionation techniques, Palade et al. then went on to isolate these

cellular components. However, the cell fractionation procedure suffered from

limitations and resulted in imperfect separation of components. A major

breakthrough was made in 1967 when a combined approach o f radiography

and an i£ vitro subcellular system was used to follow the secretion of

chymotrypsinogen. This method, along with a refined subcellular fraction

technique designed by James Jamieson, resulted in the discovery o f the

'Palade pathway'. The route of pulse-labelled enzyme was traced from its

site of synthesis on ER-attached polysomes, across the membrane and into

the lumen of the ER. Chymotrypsinogen was then followed through the ER to

the Golgi apparatus and into secretory vesicles, before the final step of

expulsion.

The Palade pathway was later verified by an independent piece of

work by Novick et al. (1981) using the genetically amenable yeast,

Sacharomyces cerevisiae. A large number of conditionally lethal mutants were

generated which were defective in the ability to secrete glycoproteins such

as invertase. The mutants were physiologically analysed at the non-permissive

temperature for growth and found to be blocked at various stages of the

secretion pathway. Furthermore, all but one of the secretion mutants

accumulated exaggerated secretory organelles. By constructing double

secretion mutants it became possible to determine the order o f secretion

events. The order was similar to that previously demonstrated by Palade. In

the same piece of work the following features of this process were

demonstrated. First, secretory proteins were glycosylated as they entered the

ER. Second, at least nine sec gene products were required to transfer

material to the Golgi apparatus. This process required energy and resulted in

further glycosylatlon o f the secretory protein. Third, at least two more

functions were required to package the almost fully glycosylated proteins into

secretory vesicles. Finally, the transportation of this bud and its fusion to

the plasma membrane required energy and a further ten gene products.

A fter these important discoveries attention was turned to the

molecular events of protein targeting. It was clearly demonstrated that

secreted proteins were initially synthesised on ER-bound polysomes suggesting

that elongation and translocation might be qoupled. It was not until 1972,

however, that a working model was presented to explain the molecular

mechanism by which secreted proteins were directed across the membrane

and into the lumen of the ER.

1 J J . The signal hypothesU

Milstein et al. (1972) discovered a slight discrepancy between the size

of IgGl produced in vitro when compared to the same immunoglobin produced

and secreted in vivo. The protein synthesized in vitro had a higher molecular

mass and exhibited an altered N-terminal amino acid sequence. From these

observations it was speculated that the alteration in the N-terminal region

might somehow be involved in the targeting o f this protein. It was postulated

that this 'leader' sequence might be acting as a signal responsible for

protein targeting.

Blobel and Dobberstein (1975) made an important breakthrough by

developing an hi vitro assay for protein secretion. By adding microsomal

vesicles to an jn vitro translation system they managed to couple protein

synthesis with its transfer across a membrane. It was demonstrated that

proteins were transferred into these microsomal vesicles and were

subsequently resistant to proteolytic degradation upon the addition o f

proteases. Proteins synthesised in the absence of vesicles were slightly larger

than those transported into microsomal vesicles. Also, if vesicles were added

after the completion of protein elongation, they were not translocated into

the vesicles. These results, as well as agreeing with those o f Milstein et al.

(1972), also demonstrated a tight coupling of protein elongation and

translocation in this system. It was also proved that the processing observed

by Milstein et al. (1972) was linked with tran$-membrane transport.

The signal hypothesis suggested that the Information for protein

translocation across membranes resided in a short 'leader' N-terminal

sequence which was removed a fter transport was complete. The result was a

shortened mature form of the protein with an altered N-terminal region.

Walter and Blobel (1980) then went on to isolate the components o f

the secretion apparatus. By salt washing rough ER membranes, a 250 kD

complex was isolated which consisted of six polypeptides. This complex was

identified as the signal recognition particle (SRP). The role of this complex

was to bind to the signal-sequence of nascent polypeptides thus preventing

further elongation (Walter and Blobel, 1981). Another protein was also

discovered which was also released from washed/protease-treated microsomes

(Meyer and Dobbersteln, 1980). This 72 kD protein restored the elongation of

the translationally arrested protein and was called the docking protein (Meyer

et al., 1982).

The early events of protein secretion across the ER membrane and

into the ER lumen are illustrated in Figure 1.1. This elaborate mechanism is

thought to exist to ensure that the secretory protein is translocated across

the ER membrane co-translationally, thus preventing it from folding into a

translocation-incompetent state. The events leading to protein export in

Gram-negative bacteria, and protein secretion in Gram-positive bacteria

(which will be discussed in later sections), appear to be similar to the Initial

steps o f secretion in eukaryotes. In both types of prokaryote, translocated

proteins are generally synthesised as precursors with N-terminal

signal-sequences. Also, some Escherichia coll proteins are known to share

sequence similarities to components o f the eukaryotic SRP complex (Bassford

et al., 1991). However, these proteins (FfH and FtsY) are not thought to be

involved in the Sec mediated export pathway in 13. coll. The Sec mediated

export pathway (also known as the general export pathway [G EP]) is

discussed in section 1.4.2. As yet, there is no evidence to suggest that a

bacterial SRP exists. Although appearing similar, the mechanisms o f export in

Gram-negative bacteria (secretion in Gram-positive bacteria) and the Initial

steps o f secretion in eukaryotes, have some differences. The main difference

might be the way that proteins are maintained In a translocation-competent state prior to translocation. The way in which prokaryotes achieve this will

Figure 1.1. Protein transport across the endoplasmic reticulum

Cytoplasm

The signal recognition particle (SRP) interacts with the N-terminal

signal-sequence emerging from the ribosome. The ribosome-nascent

polypeptide complex is directed to the endoplasmic reticulum (ER) by the

SRP and then connects with the membrane-bound docking protein ( □ ) . The

SRP is then released and protein elongation is restored. The protein is

transported through the ER membrane co-translationally and finally cleaved

by the signal peptidase (SP). The free polypeptide is released into the lumen

o f the ER.

be discussed in sections 1.4.

2

2