David I. Meyer

Secretion in yeast: translocation and glycosylation of prepro-α-factor in vitro can occur via an ATP-dependent post-translational mechanism

In an in vitro system comprising a yeast cell‐free translation system, yeast microsomes and mRNA encoding prepro‐α‐factor, the translocation of this protein across the membrane of the microsomal vesicle and its glycosylation could be uncoupled from its translation. Such post‐translational processing is dependent upon the presence of ATP in the system. It is not, however, affected by a variety of uncouplers, ionophores or inhibitors, including carbonyl cyanide m‐chlorophenyl hydrazone (CCCP), valinomycin, nigericin, dinitrophenol (DNP), potassium cyanide (KCN) or N‐ethyl maleimide (NEM). This mechanism of translocation is significant as it indicates that a protein of 18 000 daltons is capable of crossing an endoplasmic reticulum‐derived membrane post‐translationally. For the moment, this phenomenon seems to be restricted to prepro‐α‐factor in the yeast in vitro system. Neither invertase nor IgG ϰ light chain could be translocated post‐translationally in yeast, nor was such processing observed for prepro‐α‐factor in a wheat germ system supplemented with canine pancreatic microsomes.

1986: A year of new insights into how proteins cross membranes

Abstract Results published during this year indicate that many secretory and membrane proteins are capable of being translocated across ER-derived membranes when fully synthesized. Although for years a bone of contention, the distinction between co- and post-translational translocation is rapidly becoming irrelevant. New data, based on post-translational analyses, indicate that an essential feature of translocation may have to do with preventing the folding of the protein into a tertiary structure approximating that of the mature protein. This review focuses on examples of post-translational translocation that support such a hypothesis.

Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins.

Ribophorins I and II represent proteins that are postulated to be involved in ribosome binding. They are abundant, highly‐conserved glycoproteins located exclusively in the membranes of the rough endoplasmic reticulum. As the first step in the further characterization of the structure and function of these proteins, we have isolated and sequenced full‐length human cDNA clones encoding ribophorins I and II using probes derived from a human liver expression library cloned into pEX1. The authenticity of the clones was verified by overlaps in the protein sequence of N‐terminal and several internal fragments of canine pancreatic ribophorins I and II. The cDNA clones hybridize to mRNA species of 2.5 kb in length, and encode polypeptides of 68.5 and 69.3 kd, respectively. Primary sequence analysis, coupled with biochemical studies on the topology, indicates that both ribophorins are largely luminally disposed, spanning the membrane once and having 150 and 70 amino acid long cytoplasmically disposed C termini, respectively. Both are synthesized as precursors having cleavable signal sequences of 23 (ribophorin I) and 22 (ribophorin II) amino acids. The topology suggested by the primary structure has been confirmed biochemically using proteolytic enzymes and anti‐ribophorin antibodies. Proteolysis of intact microsomes with a variety of enzymes resulted in a reduction in the apparent mol. wt of ribophorin I that would correspond to a loss of its 150‐amino acid cytoplasmic tail. In the case of ribophorin II, it is completely resistant to such proteolysis which is consistent with its luminal disposition and fairly hydrophobic C terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of the p62 subunit of dynactin with Arp1 and the cortical actin cytoskeleton

Targeting of the minus-end directed microtubule motor cytoplasmic dynein to a wide array of intracellular substrates appears to be mediated by an accessory factor known as dynactin [1-4]. Dynactin is a multi-subunit complex that contains a short actin-related protein 1 (Arp 1) filament with capZ at the barbed end and p62 at the pointed end [5]. The location of the p62 subunit and the proposed role for dynactin as a multifunctional targeting complex raise the possibility of a dual role for p62 in dynein targeting and in Arp1 pointed-end capping. In order to gain further insight into the role of p62 in dynactin function, we have cloned cDNAs that encode two full-length isoforms of the protein from rat brain. We found that p62 is homologous to the nuclear migration protein Ropy-2 from Neurospora [6]; both proteins contain a zinc-binding motif that resembles the LIM domain of several other cytoskeletal proteins [7]. Overexpression of p62 in cultured mammalian cells revealed colocalization with cortical actin, stress fibers, and focal adhesion sites, sites of potential interaction between microtubules and the cell cortex [8,9]. The p62 protein also colocalized with polymers of overexpressed wild-type or barbed-end-mutant Arp1, but not with a pointed-end mutant. Deletion of the LIM domain abolished targeting of p62 to focal-adhesion sites but did not interfere with binding of p62 to actin or Arp1. These data implicate p62 in Arp1 pointed-end binding and suggest additional roles in linking dynein and dynactin to the cortical cytoskeleton.

An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 × 10(−5) M. ATP transport into ER vesicles was specifically inhibited by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3′‐O‐(4‐benzoyl)benzoyl‐ATP (Bz2‐ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2‐ATP blocked protein translocation in vitro measured co‐ as well as post‐translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS‐treated membranes. These results demonstrate that translocation requires a DIDS and Bz2‐ATP‐sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.

Secretion in yeast: structural features influencing the post-translational translocation of prepro-alpha-factor in vitro.

In vitro, efficient translocation and glycosylation of the precursor of yeast alpha‐factor can take place post‐translationally. This property of prepro‐alpha‐factor appears to be unique as it could not be extended to other yeast protein precursors such as preinvertase or preprocarboxypeptidase Y. In order to determine if specific domains of prepro‐alpha‐factor were involved in post‐translational translocation, we carried out a series of experiments in which major domains were either deleted or fused onto reporter proteins. Fusion of various domains of prepro‐alpha‐factor onto the reporter protein alpha‐globin did not allow post‐translational translocation to occur in the yeast in vitro system. Prepro‐alpha‐factor retained its ability to be post‐translationally translocated when parts or all of the pro region were deleted. Removal of the C‐terminal repeats containing mature alpha‐factor had the most profound influence as post‐translational translocation decreased in proportion to the number of repeats deleted. Taken together, these results suggest that efficient post‐translational translocation requires a signal sequence and the four C‐terminal repeats. There does not however, appear to be specific information contained within the C‐terminus, as their presence in fusion did not enable the post‐translational translocation of reporter proteins. Lastly, the ability to post‐translationally translocate radiochemically pure prepro‐alpha‐factor that had been isolated by immuno‐affinity chromatography required the addition of a yeast lysate fraction. Moreover, post‐translational translocation is a function of the microsomal membrane of yeast microsomes and not of a factor peculiar to the yeast lysate, as reticulocyte lysate supported this as well.

The chromaffin granule surface: the presence of actin and the nature of its interaction with the membrane

The involvement of the cytoskeleton in stimulussecretion coupling has been implied through several lines of evidence (reviewed in [ 1] ). Secretion can be inhibited by drugs known to disrupt cytoskeletal function [2], microfilamentous structures have been detected morphologically in secretory cells [3] and actin has been identified as a prominent component in such tissues [4,5]. Microfilaments and/or actin have been found in association with isolated secretory granules [6] and purified actin has been shown capable of ‘reassociating’ with membranes of set, .-tory granules [7,8]. There has been a good deal of controversy surrounding the question of whether or not secretory granules from adrenal medulla (chromaffin granules) possess endogenous actin. Datahas been presented [9] suggesting that actin is not present in isolated granules, while earlier reports have suggested that there is actin that associates with granule membranes [7]. While the reports in [7,9] relied solely on data obtained by gel electrophoresis (and fingerprint analysis [7] but not on granule membrane fractions), we used immunoprecipitation with monospecific antibodies to actin to test the presence of actin on granule membranes. Due to the lack of information on secretory granule-

Signal recognition particle (SRP) does not mediate a translational arrest of nascent secretory proteins in mammalian cell-free systems

The ability of the signal recognition particle (SRP) to induce translational arrests in wheat germ, reticulocyte and HeLa cell‐free translation systems was examined. In accordance with published data, SRP caused a complete arrest of secretory protein (IgG light chain) translation in wheat germ. In contrast, SRP had no effect on translation in either reticulocyte or HeLa cell lysates, even at 5‐fold higher SRP levels than needed for complete arrest in wheat germ. The existence of a “docking‐protein‐like” releasing activity was ruled out, in the case of reticulocyte lysate, by experiments in which reticulocyte subfractions were added to blocked translations in wheat germ. In the absence of additional evidence to the contrary, it seems as if the translational arrest is peculiar to the wheat germ cell‐free system.

Transfer of secretory proteins through the membrane of the endoplasmic reticulum.

Publisher Summary Secretory proteins are synthesized on ribosomes located in the cytoplasm and are released from the cell as a fairly homogeneous, highly concentrated population of molecules. Secretory proteins can be localized in the lumen of the endoplasmic reticulum (ER) very early in their existence. Translocation of secretory proteins from the cytoplasmic to the luminal side of the rough ER membrane is an essential feature of the transport of these molecules. The information for the association of the mRNAs encoding secretory proteins to the membrane is contained within secretory protein mRNAs itself. mRNA encoding a secretory protein contains the signal for its ultimate association with the membrane. All secretory protein mRNAs encode a transient N-terminal peptide. This short amino acid sequence provides a signal, which results in the formation of membrane-bound polysomes and, ultimately, the translocation across the membrane. This chapter presents the results of studies, which deal with various individual aspects of protein translocation. It also describes signal sequences, signal recognition particle, docking protein, ribosome binding, signal peptidase, and the cotranslational covalent modifications, which are carried out in the rough ER.

Induction of secretory pathway components in yeast is associated with increased stability of their mRNA

The overexpression of certain membrane proteins is accompanied by a striking proliferation of intracellular membranes. One of the best characterized inducers of membrane proliferation is the 180-kD mammalian ribosome receptor (p180), whose expression in yeast results in increases in levels of mRNAs encoding proteins that function in the secretory pathway, and an elevation in the cells ability to secrete proteins. In this study we demonstrate that neither the unfolded protein response nor increased transcription accounts for membrane proliferation or the observed increase in secretory pathway mRNAs. Rather, p180-induced up-regulation of certain secretory pathway transcripts is due to a p180-mediated increase in the longevity of these mRNA species, as determined by measurements of transcriptional activity and specific mRNA turnover. Moreover, we show that the longevity of mRNA in general is substantially promoted through the process of its targeting to the membrane of the endoplasmic reticulum. With respect to the terminal differentiation of secretory tissues, results from this model system provide insights into how the expression of a single protein, p180, could result in substantial morphological and functional changes.