The analysis of protein sorting signals responsible for the retention of reticuloplasmins (RPLs), a group of soluble proteins that reside in the lumen of the endoplasmic reticulum (ER), has revealed a structural similarity between mammalian and plant ER retention signals. We present evidence that the corresponding epitope is conserved in a vast family of soluble ER resident proteins. Microsequences of RPL60 and RPL90, two abundant members of this family, show high sequence similarity with mammalian calreticulin and endoplasmin. RPL60/calreticulin cofractionates and costains with the lumenal binding protein (BiP). Both proteins were detected in the nuclear envelope and the ER, and in mitotic cells in association with the spindle apparatus and the phragmoplast. Immunoprecipitation of proteins from in vivo-labeled cells demonstrated that RPL60/calreticulin is associated with other polypeptides in a stress- and ATP-dependent fashion. RPL60/calreticulin transcript levels increased rapidly in abundance during the proliferation of the secretory apparatus and the onset of hydrolase secretion in gibberellic acid-treated barley aleurone cells. This induction profile is identical to that of the well-characterized ER chaperones BiP and endoplasmin. However, expression patterns in response to different stress conditions as well as tissue-specific expression patterns indicate that these genes are differentially regulated and may not act in concert.

The analysis of protein sorting signals responsible for the retention of reticuloplasmins (RPLs), a group of soluble proteins that reside in the lumen of the endoplasmic reticulum (ER), has revealed a structural similarity between mammalian and plant ER retention signals. We present evidence that the corresponding epitope is conserved in a vast family of soluble ER resident proteins. Microsequences of RPL60 and RPL90, two abundant members of this family, show high sequence similarity with mammalian calreticulin and endoplasmin. RPL60/calreticulin cofractionates and costains with the lumenal binding protein (BiP). Both proteins were detected in the nuclear envelope and the ER, and in mitotic cells in association with the spindle apparatus and the phragmoplast. Immunoprecipitation of proteins from in vivo-labeled cells demonstrated that RPL60/calreticulin is associated with other polypeptides in a stress- and ATP-dependent fashion. RPL60/calreticulin transcript levels increased rapidly in abundance during the proliferation of the secretory apparatus and the onset of hydrolase secretion in gibberellic acid-treated barley aleurone cells. This induction profile is identical to that of the well-characterized ER chaperones BiP and endoplasmin. However, expression patterns in response to different stress conditions as well as tissue-specific expression patterns indicate that these genes are differentially regulated and may not act in concert.

No abstract

In this paper we show, that the in vivo methylation of the elongation factor Tu from Eschericliia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine.We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site I1 is. Although the apparent affinity of tRNA for site I1 remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated.A possible physiological implication of this phenomenon is discussed.In living organisms, many cellular components become selectively modified during their lifetime. The N-methylation of proteins is an example of such modifications. Very little is known about the functional role of protein methylation. The elongation factor Tu (EF-Tu) from Escherichia coli, an abundant protein which plays an important role during protein biosynthesis [l -31, is an example of this. As yet, it is the only non-ribosomal protein of E. coli reported to become Nmethylated [4]. The N-methylation of EF-Tu only occurs at one specific position, i.e. Lys-56, and has been suggested to depend upon the growth phase of the bacterium [4]. No data supporting this assumption, however, have been published so far. Although EF-Tu methylation could also be obtained in an in vitro system [5], in fact nothing is known about the structural or functional effect(s) of the methylation upon EFTu. Apart from the blocked N-terminus (N-acetyl-serine), no other post-translational modifications of EF-Tu have been found.Although progress has been made in the elucidation of the tertiary structure of EF-Tu, crystals suitable for X-ray diffraction studies are only obtained upon mild tryptic cleavage of the protein. Trypsin excises the fragment 44-58, which includes Lys-56 [6]. The position of Lys-56 can therefore not yet be indicated in the preliminary model of the tertiary structure of EF-Tu [7, 81.

SummaryWe have identi®ed the major endo-b-1,4-xylanase (XYN-1) in the aleurone of germinating barley grain, and show that it is expressed as a precursor of M r 61 500 with both N-and C-terminal propeptides. XYN-1 is synthesized as an inactive enzyme in the cytoplasm, and only becomes active at a late stage of germination when the aleurone ceases to secrete hydrolases. A series of processing steps, mediated in part by aleurone cysteine endoproteases, yields a mature active enzyme of M r 34 000. Processing and extracellular release of the mature enzyme coincide with the programmed cell death (PCD)-regulated disintegration of aleurone cells. We discuss the signi®cance of delayed aleurone cell-wall degradation by endoxylanases in relation to the secretory capacity of the aleurone, and propose a novel role for aleurone PCD in facilitating the export of hydrolases.