JOHN ARMSTRONG PROTEIN TRANSPORT

Breaking the topological dogma Some proteins can be delivered to the yeast

vacuole by mechanisms that do not involve the usual pathway through the endoplasmic reticulum.

The study of protein transport between membrane com- partments of the eukaryotic cell is unified by the princi- ple, rarely stated but widely accepted, of conservation of topology. Thus, as all the membranes involved in the se- cretory and endocytotic pathways are connected, either physically or by vesicle trafhc, every site in the cell is ei- ther ‘in’ or ‘out’. (Peroxisomes, mitochondria and related organelles will not be included in the following discus- sion.) For example, the lumen of the endoplasmic retic- ulum (ER), the inside of the lysosome and the space be- tween the inner and outer nuclear envelope are all ‘out’, whereas the cytosol and the inside of the nucleus are both ‘in’. A protein’s relationship to this topological bar- rier is defined at its birth. ‘In’ proteins are synthesized in the cytosol, whereas nascent ‘out’ and transmembrane proteins are synthesized on ribosomes associated,with the ER, and then translocated into or through the ER [I]. After its synthesis, no further membrane translocation of a protein is possible, so its topological status is now lixed. It may only be altered by the breakdown of a membrane, for example on cell death.

Exceptions to this rule have been reported, but these generally involve short peptides rather than full pro- teins. For example, the yeast a-type mating factor and a variety of cytosolic peptides that bind to the major histo- compatibility complex ((MHC) class I molecule seem to be translocated by specific pumps that act post-transla- tionally [2]. In the latter case, it had been thought that whole proteins might first be translocated and then pro- teolysed within the ER but the recent discovery of genes within the MHC complex that are homologous to cyto- plasmic ‘proteasome’ components 131 is further evidence

that degradation of proteins to peptides occurs in the cy- tosol. A different kind of exception is the discovery that Succburonzyces cerevzkiae can just about survive without a functional signal recognition particle (SRP), a part of the machinery for targeting to the ER [4]. However, in this case it may be that the role of the SRP is partly re- placed by heat-shock proteins, and the actual machinery for translocation into the ER is the same as usual. Two rather more convincing exceptions to the rule have now been observed. Both involve the delivery of pro- teins to the yeast vacuole (the approximate equivalent of the lysosome in animal cells). The first case, reported by Chiang and Schekman [ 51, concerns the gluconeogenic enzyme, fructose 16bisphosphatase (FBPase), which is involved in the synthesis of glucose from non-carbohy- drate substrates. When yeast were switched from acetate to glucose as carbon source, FBPase, which is normally cytoplasmic, was rapidly degraded by proteases within the vacuole. When the change of medium was performed with yeast that are defective in the relevant protease, in- tact FBPase was delivered into the vacuole. This was demonstrated by both cell fractionation and immuno- fluorescence techniques. Temperature-sensitive mutants blocked in the early stages of secretion did not degrade the protein. Thus, surprisingly, the secretory pathway is necessary for degradation. Yet FBPase has no ER signal sequence and did not accumulate within membranes in the secretion-blocked cells. How, then is FBPase targeted in yeast grown on glucose? Chiang and Schekman [5] postulate, that in response to glucose, a transport protein or ‘translocator’ is synthe- sized at the ER and delivered to the vacuole by the con-

Fig. 1. The unusual pathway by which the cytosolic FBPase (red) enters the vacuole. The putative FBPase transloca- tor (blue) requires the normal secretory pathway for transport to the vacuole.

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ventional pathway. There it is activated and causes selec- tive uptake of FBPase (Fig. 1). (Mammalian lysosomes also exhibit regulated uptake of proteins from the cytosol [61 but, in the absence of genetic tools, the mechanisms involved will be harder to study.) These processes could represent a new form of protein translocation. Alterna- tively, uptake could occur by some form of autophagy, in which proteins are ‘endocytosed’ into the vacuole and then exposed to lprotease by lysis of the internalized membrane. In this case, of course, the topological dogma would remain unbreached.

The second, and perhaps even more striking, example of an unusual uptake mechanism is that of u-mannosi- dase. Unlike FBPase, this enzyme’s normal site of ac- tion is within the vacuole. However, on cloning its gene, Yoshihisa and Anraku [7,8] found no obvious ER sig- nal sequence. What is even more strange is that the en- zyme is not glycosylated, in spite of possessing seven po- tential sites for N-glycosylation. There is nothing intrin- sically wrong with these sites because when the N-ter- minal part of the enzyme was replaced with that of car boxypeptidase Y, a conventionally-routed vacuolar pro- tein, the mannosidase portion became glycosylated. In the reciprocal expleriment, in which a truncated version of invertase was attached to the C-terminus of mmamiosi- dase, an unglycosylated vacuolar protein was produced. In contrast to FBPase, delivery of a-mannosidase was not inhibited by mutations in the secretory pathway.

These elegant experiments with a-mannosidase provide the first clear example of a large eukaryotic protein reach- ing its destination in the secretory or endocytotic system by a mechanism that is independent of the ER transloca- tion apparatus. Why should the yeast cell devise another delivery system when it already has at least one perfectly good one? A clue may lie in the enzyme’s degradative function: if the mannosidase entered the secretory path way, it would be let loose on the newly-synthesized gly- coproteins in the ER and Golgi. In the case of vacuolar

346 @ 1991 Current Biology

proteases, which present the same threat, the problem is averted by their synthesis as ‘pro’-forms that are later activated by either the low pH of the vacuole or other proteolytic enzymes.

The mechanisms involved in the unusual transport of FBPase and a-mannosidase remain unknown but, being in yeast, should be amenable to genetic analysis, Mean- while, the central dogma of membrane traflic seems to have gone the way of the other dogmas of molecular bi- ology: still essentially true, but prone to some interesting exceptions.

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SCHATZ G: Protein translocation machine in yeast. CUYT Biol 1991, h4z-44. SAMBROOK JF: Pumping peptides. Cur-r Eli01 1991, 1:57-58.

GL~NE R, Pours SH, BECK S, KELLY A, KERR L-A, TF~OWSDALE J: A proteasome-related gene between the two ABC transporter loci in the class II region of the human MHC. Nature 1991, 353:357-360. HANN B, WALTER P: The signal recognition particle of S. cere-

visiue. Cell 1391, 67:131-144. CHLWG H-L, SCHEKMAN R Regulated import and degradation of a cytosolic protein in the yeast vacuole. Nature 1991, 350:313-318.

DICE JP: Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trend Biocbem Sci 1990, 15305-309.

YOSHMISA T, ANRAKU Y: Nucleotide sequence of AM.%, the structure gene of vacuolar a-mannosidase of Saccha- romyces cerevisiae. Bitiem Biopbys Res Commun 1989, 163(2):908-915.

YOSHIHISA T, ANRAKU Y: A novel pathway of import of CL- man- nosidase,. a marker enzyme of vacuolar membrane, in Sac- charomyces cerevisiae. J Biol Cbem WO, 265:22318-22425.

John Armstrong, Membrane Molecular Biology Iabora- tory, Imperial Cancer Research Fund, PO Box 123, Lin- coln’s Inn Fields, London WC2A 3PX, UK.

cc That’s life

Scientific research can be a dull grey way of spending one’s life, and it is small wonder that scientists are such a boring lot. Peter Williams was a blazing exception who advanced biology not, as with his intellect he could well have done, by his own research and findings but by encouraging and helping his colleagues, civilizing them in the process.

From an obituary in The Independent, 12 April