Translation rate of human tyrosinase determines its N-linked glycosylation level.

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or seven N-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential cotranslational maturation events.

Selective ubiquitination of calmodulin by UBC4 and a putative ubiquitin protein ligase (E3) from Saccharomyces cerevisiae.

A putative ubiquitin protein ligase (E3-CaM) which cooperates with UBC4 in selectively ubiquitinating calmodulin has been partially purified from Saccharomyces cerevisiae. Ca2+ was required for this activity and monoubiquitinated calmodulin was the main product of the reaction. The apparent Km of E3-CaM for calmodulin was approximately 1 microM which is of the same order of magnitude as the concentration of calmodulin in yeast cells. Proteins which are good substrates for other E3s (E3 alpha or E3-R) were not ubiquitinated by E3-CaM. Lower but significant activities of E3-CaM were observed when UBC1 replaced UBC4.

RAD6 gene product of Saccharomyces cerevisiae requires a putative ubiquitin protein ligase (E3) for the ubiquitination of certain proteins.

The RAD6 (UBC2) gene of Saccharomyces cerevisiae which is involved in DNA repair, induced mutagenesis, and sporulation, encodes a ubiquitin-conjugating enzyme (E2). Since the RAD6 gene product can transfer ubiquitin directly to histones in vitro without the participation of a ubiquitin protein ligase (E3), it has been suggested that in vivo it also acts by the unassisted conjugation of ubiquitin to histones or to other target proteins. Here we show that the RAD6 protein can ligate ubiquitin in vitro to a hitherto unknown set of exogenous target proteins (alpha-, beta-, and kappa-casein and beta-lactoglobulin) when supplemented by a putative ubiquitin protein ligase (E3-R) from S. cerevisiae. RAD6 supplemented with E3-R ligates 1 or, sometimes, 2 ubiquitin molecules to the target protein molecule. UBC3 (CDC34) protein in the presence of E3-R has barely detectable activity on the non-histone substrates. Other ubiquitin-conjugating enzymes tested (products of the UBC1 and UBC4 genes) do not cooperate with E3-R in conjugating ubiquitin to the same substrates. Thus, E3-R apparently interacts selectively with RAD6 protein. These findings suggest that some of the in vivo activities of the RAD6 gene may involve E3-R.

Effect of stress on protein degradation: role of the ubiquitin system.

Many factors which induce the stress response (heat shock protein synthesis) in eukaryotes also cause the formation of aberrant proteins. Such aberrant proteins are usually rapidly and selectively degraded in cells. Temperature step-up accelerates the degradation of a subset of normally stable proteins. This effect is transient and is confined to a narrow range of heat shock temperatures above which proteolysis is inhibited. The time course and extent of proteolysis elicited by a mild heat shock is consistent with data on the thermal transitions of cellular proteins. Biochemical and genetic evidence strongly supports the view that the ubiquitin system is primarily responsible for heat- or stress-damaged protein degradation in eukaryotic cells. It still remains to be determined how stress-damaged proteins are recognized by the ubiquitin system and selected for degradation. Ubiquitin-protein ligases (E3's) which attach multi-ubiquitin chains to proteins are thought to be responsible for the selection of proteins for degradation. Several species of E3 have recently been characterized. However, none of the known E3's seems to fulfil the role of selecting aberrant proteins for breakdown. Heat shock proteins which are thought to repair unfolded or misfolded proteins probably have a complementary function to the ubiquitin system which destroys damage proteins. The relationship between the ubiquitin system and the regulation of heat shock protein synthesis, which is still not understood, is discussed.

Ubiquitin in Chlamydomonas reinhardii. Distribution in the cell and effect of heat shock and photoinhibition on its conjugate pattern.

Ubiquitin, a highly conserved 76-amino-acid protein, is involved in the response of many types of eukaryotic cells to stress but little is known about its role in lower plants. In the present study we have investigated the distribution of ubiquitin in the unicellular alga Chlamydomonas reinhardii as well as the effect of heat and light stress on its conjugation to cellular proteins. Immunoelectron microscopy shows that ubiquitin is located in the chloroplast, nucleus, cytoplasm, pyrenoid and on the plasma membrane. The location of ubiquitin within chloroplasts has not been observed previously. In immunoblots of whole cell extracts with an antibody to ubiquitin a prominent conjugate band with an apparent molecular mass of 29 kDa and a broad region of high-molecular-mass conjugates (apparent molecular mass greater than 45 kDa) were observed. Exposure of cells to a 41.5 degrees C heat shock in both the dark and light caused the disappearance of the 29-kDa conjugate and an increase in the high-molecular-mass conjugates. After step down to 25 degrees C the 29-kDa conjugate reappeared while the levels of high-molecular-mass conjugates decreased. In light, the recovery of the 29-kDa band was more rapid than in the dark. Photoinhibition alters the ubiquitin conjugation pattern similarly to heat shock, but to a lesser degree. These observations imply that, in Chlamydomonas, ubiquitin has a role in the chloroplast and in the response to heat and light stress.

A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1.

The effect of restrictive temperature on ubiquitin conjugation activity has been studied in cells of ts20, a temperature-sensitive cell cycle mutant of the Chinese hamster cell line E36. Ts20 is arrested in early G2 phase at nonpermissive temperature. Immunoblotting with antibodies to ubiquitin conjugates shows that conjugates disappear rapidly at restrictive temperatures in ts20 mutant but not in wild type E36 cells. The incorporation of 125I-ubiquitin into permeabilized ts20 cells is temperature-sensitive. Addition of extracts of another G2 phase mutant, FM3A ts85, with a temperature-sensitive ubiquitin activation enzyme (E1), to permeabilized ts20 cells at restrictive temperatures fails to complement their ubiquitin ligation activity. This indicates that the lesions in the two mutants are similar. Purified E1 from reticulocytes restores the conjugation activity of heat-inactivated permeabilized ts20 cells. Ubiquitin conjugation activity of cell-free extracts of ts20 cells was temperature-sensitive and could be restored by adding purified reticulocyte E1. Purified reticulocyte E2 or E3, on the other hand, did not restore the ubiquitin conjugation activity of heat-treated ts20 extracts. These results are consistent with the conclusion that ts20 has temperature-sensitive ubiquitin-activating enzyme (E1). The fact that two E1 mutants (ts20 and ts85) derived from different cell lines are arrested at the S/G2 boundary at restrictive temperatures strongly indicates that ubiquitin ligation is necessary for passage through this part of the cell cycle. The temperature thresholds of heat shock protein synthesis of ts20 and wild type E36 cells were identical. The implications of these findings with respect to a suggested role of ubiquitin in coupling between protein denaturation and the heat shock response are discussed.

Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.

Exposure of cultured rat hepatoma (HTC) cells to a 43 degrees C heat shock transiently accelerates the degradation of the long-lived fraction of cellular proteins. The rapid phase of proteolysis which lasts approximately 2 h after temperature step-up is followed by a slower phase of proteolysis. During the first 2 h after temperature step-up there is a wave of ubiquitin conjugation to cellular proteins which is accompanied by a fall in ubiquitin and ubiquitinated histone 2A (uH2A) levels. Upon continued incubation at 43 degrees C the levels of ubiquitin conjugates fall with a corresponding increase of ubiquitin and uH2A to initial levels. The burst of protein degradation and ubiquitin conjugation after temperature step-up is not affected by the inhibition of heat shock protein synthesis. Cells of the FM3A ts85 mutant, which have a thermolabile ubiquitin activating enzyme (E1), do not accelerate protein degradation in response to a 43 degrees C heat shock, whereas wild-type FM3A mouse cells do. This observation indicates that the ubiquitin system is involved in the degradation of heat-denatured proteins. Sequential temperature jump experiments show that the extent of proteolysis at temperatures up to 43 degrees C is related to the final temperature and not to the number of steps taken to attain it. Temperature step-up to 45 degrees C causes the inhibition of intracellular proteolysis. We propose the following explanation of the above observations. Heat shock causes the conformational change or denaturation of a subset of proteins stable at normal temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

Conjugation of [125I]ubiquitin to cellular proteins in permeabilized mammalian cells: comparison of mitotic and interphase cells.

[125I]Ubiquitin introduced into permeabilized hepatoma tissue culture (HTC) cells rapidly forms conjugates with endogenous proteins. A characteristic pattern of low mol. wt conjugates is obtained which includes the ubiquitinated histone, uH2A, and unknown molecular species with MrS of 14, 23, 26 (two bands) and 29 kd. A broad spectrum of higher mol. wt conjugates is also produced. The formation of all conjugates is absolutely dependent on ATP, and upon depletion of ATP they are rapidly broken down. The 14, 23 and 29 kd species are found in all subcellular fractions examined. uH2A is located exclusively in the nuclear fraction. The pair of 26 kd bands is specifically associated with the ribosome fraction. A considerable percentage of the higher mol. wt conjugates sediments with the small particle (100,000 g) fraction in the ultracentrifuge but is solubilized with deoxycholate, indicating that there are many membrane-associated conjugates. The pattern of ubiquitin conjugation in interphase and metaphase cells was compared. The incorporation of ubiquitin into uH2A was markedly reduced in metaphase cells whereas its incorporation into other low mol. wt conjugates and into high mol. wt conjugates was affected slightly, if at all. This shows that the known decrease of uH2A levels in metaphase is due to a specific effect on histone ubiquitination and not to a general decrease in ubiquitination activity or increase of isopeptidase activity. Changes in the levels of uH2A during mitosis measured by immunoblotting were similar to those estimated in permeabilized cells. These experiments indicate that permeabilized cells provide a useful approach to the study of rapidly turning over ubiquitin conjugates in mammalian cells.