Hugh R.B. Pelham

An hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein

We have characterized a cDNA clone that encodes a protein related to the 70 kd heat shock protein, but is expressed in normal rat liver. This protein has a hydrophobic leader and is secreted into the endoplasmic reticulum. We show that it is identical with two previously described proteins: GRP78, whose synthesis is induced by glucose starvation, and BiP, which is found bound to immunoglobulin heavy chains in pre-B cells. This protein, which is abundant in antibody-secreting cells, can be released from heavy chains by ATP, a reaction analogous to the release of hsp70 from heat shocked nuclear structures. We propose a specific role for this protein in the assembly of secreted and membrane-bound proteins.

Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation

Heat shock promoters contain one or more binding sites for a specific heat shock factor (HSF). We report the cloning and sequence of the gene encoding yeast HSF, and demonstrate that HSF is required for growth at normal temperatures (15 degrees C-30 degrees C). The activity of a promoter containing a synthetic HSF binding site varies over a 200-fold range between 15 degrees C and 39 degrees C (heat shock). This change in activity is accompanied by multiple changes in the phosphorylation state of HSF, but all forms of HSF are able to bind DNA. We propose that the expression of heat shock genes in yeast is modulated by phosphorylation of DNA-bound HSF, and that this leads to a more efficient interaction of the factor with other components of the transcriptional machinery.

The retention signal for soluble proteins of the endoplasmic reticulum

The lumen of the endoplasmic reticulum (ER) contains a number of soluble proteins, many of which help the maturation of newly synthesized secretory proteins. Retention of these resident proteins in the ER is dependent on a carboxy-terminal signal, which in animal cells is usually Lys-Asp-Glu-Leu (KDEL). This signal is thought to be recognized by a membrane-bound receptor that continually retrieves the proteins from a later compartment of the secretory pathway and returns them to the ER.

ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway.

Resident proteins of the ER lumen carry a specific tetrapeptide signal (KDEL or HDEL) that prevents their secretion. We have previously described the isolation of yeast mutants that fail to retain such resident proteins within the cell. Here we describe ERD2, a gene required for retention. It encodes a 26 kd integral membrane protein whose abundance determines the efficiency and capacity of the retention system. Reduced expression of ERD2 leads to secretion of proteins bearing the HDEL signal, whereas overexpression of ERD2 improves retention both in wild-type cells and in other mutants. These results are consistent with other evidence that ERD2 encodes the HDEL receptor (see accompanying paper). The gene is also required, perhaps indirectly, for normal protein transport through the Golgi, and hence for growth. We discuss possible roles for ERD2 in the secretory pathway.

Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum

Resident luminal endoplasmic reticulum (ER) proteins carry a targeting signal (usually KDEL in animal cells) that allows their retrieval from later stages of the secretory pathway. In yeast, the receptor that promotes this selective retrograde transport has been identified as the product of the ERD2 gene. We describe here the properties of a human homolog of this protein (hERD2). Overproduction of hERD2 improves retention of a protein with a weakly recognized variant signal (DDEL). Moreover, overexpression of KDEL or DDEL ligands causes a redistribution of hERD2 from the Golgi apparatus to the ER. Mutation of hERD2 alters the ligand specificity of this effect, implying that it interacts directly with the retained proteins. Ligand control of receptor movement may limit retrograde flow and thus minimize fruitless recycling of secretory proteins.

Sorting of proteins into multivesicular bodies: ubiquitin‐dependent and ‐independent targeting

Yeast endosomes, like those in animal cells, invaginate their membranes to form internal vesicles. The resulting multivesicular bodies fuse with the vacuole, the lysosome equivalent, delivering the internal vesicles for degradation. We have partially purified internal vesicles and analysed their content. Besides the known component carboxypeptidase S (Cps1p), we identified a polyphosphatase (Phm5p), a presumptive haem oxygenase (Ylr205p/Hmx1p) and a protein of unknown function (Yjl151p/Sna3p). All are membrane proteins, and appear to be cargo molecules rather than part of the vesicle‐forming machinery. We show that both Phm5p and Cps1p are ubiquitylated, and that in a doa4 mutant, which has reduced levels of free ubiquitin, Cps1p, Phm5p and Hmx1p are mis‐sorted to the vacuolar membrane. Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain. In contrast, Sna3p enters internal vesicles in a ubiquitin‐independent manner. Thus, ubiquitin acts as a signal for the partitioning of some, but not all, membrane proteins into invaginating endosomal vesicles.

Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling

Many cells show a polarized distribution of some plasma membrane proteins, which may be maintained either by a diffusion barrier or kinetically: as first demonstrated in fibroblasts, locally exocytosed proteins will remain polarized if they are endocytosed and recycled before they can diffuse to equilibrium. In yeast, actin cables direct exocytosis to the bud and to the tips of polarized mating intermediates termed shmoos. A septin ring at the bud neck retains some proteins, but shmoos lack this. Here, we show that the exocytic SNARE Snc1 is kinetically polarized. It is concentrated at bud and shmoo tips, and this requires its endocytosis. Kinetic polarization is possible in these small cells because proteins diffuse much more slowly in the yeast plasma membrane than would be expected from measurements in animal cells. Slow diffusion requires neither the cell wall nor polymerized actin, but it is affected in the ergosterol synthesis mutant erg6. Other proteins also require endocytosis for efficient polarization, and the plasma membrane SNARE Sso1 can be polarized merely by appending an endocytic signal. Thus, despite their small size, yeast cells can use localized exocytosis and endocytic recycling as a simple mechanism to maintain polarity.

An ecdysone response element in the Drosophila hsp27 promoter.

It has previously been shown that a region of ˜100 bp in the Drosophila hsp27 promoter is sufficient to confer ecdysone inducibility on a heterologous gene. We now show, using binding and DNase I footprinting assays, that a 23‐bp hyphenated dyad within this sequence forms a protein‐binding site, and that this is sufficient for inducibility. The sequence shows partial homology with mammalian steroid receptor binding sites. UV crosslinking identifies an 80‐ to 90‐kd protein that binds specifically to this sequence and is thus a candidate for the ecdysone receptor.

Two syntaxin homologues in the TGN/endosomal system of yeast

Intracellular membrane traffic is thought to be regulated in part by SNAREs, integral membrane proteins on transport vesicles (v‐SNAREs) and target organelles (t‐SNAREs) that bind to each other and mediate bilayer fusion. All known SNARE‐mediated fusion events involve a member of the syntaxin family of t‐SNAREs. Sequence comparisons identify eight such proteins encoded in the yeast genome, of which six have been characterized. We describe here the remaining two, Tlg1p and Tlg2p. These have the expected biochemical properties of t‐SNAREs, and are located in separable compartments which correspond to a putative early endosome and the yeast equivalent of the TGN, respectively. They co‐precipitate with the v‐SNARE Vti1p, which is implicated in Golgi–endosome traffic and, remarkably, binds to five different syntaxins. Tlg1p also binds the plasma membrane v‐SNARE Snc1p. Both Tlg1p and Tlg2p are required for efficient endocytosis and to maintain normal levels of TGN proteins. However, neither is required for intra‐Golgi traffic. Since no further syntaxins have been identified in yeast, this implies that the Golgi apparatus can function with a single syntaxin, Sed5p.

Purification and characterization of a heat-shock element binding protein from yeast

The promoters of heat shock genes are activated when cells are stressed. Activation is dependent on a specific DNA sequence, the heat‐shock element (HSE). We describe the purification to homogeneity of an HSE‐binding protein from yeast (Saccharomyces cerevisiae), using sequential chromatography of whole cell extracts on heparin‐agarose, calf thymus DNA‐Sepharose and an affinity column consisting of a repetitive synthetic HSE sequence coupled to Sepharose. The protein runs as a closely spaced doublet of approximately 150 kd on SDS‐polyacrylamide gels; mild proteolysis generates a stable 70‐kd fragment which retains DNA binding activity. The relative affinities of the protein for a range of variant HSE sequences correlates with the ability of these sequences to support heat‐inducible transcription in vivo, suggesting that this polypeptide is involved in the activation of heat‐shock promoters. However, the protein was purified from unshocked yeast, and may therefore represent an unactivated form of heat‐shock transcription factor. Study of the purified protein should help to define the mechanistic basis of the heat‐shock response.