Richard G. Kulka

Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae

Combinations of different ubiquitin‐conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C‐terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C‐terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C‐terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes.

Degradation Signals Recognized by the Ubc6p-Ubc7p Ubiquitin-Conjugating Enzyme Pair

ABSTRACT Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of β-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Δ and sec61-2) did not inhibit the degradation of the β-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p.

Fusion of human erythrocyte ghosts promoted by the combined action of calcium and phosphate ions.

CALCIUM ions are important in almost all membrane fusion systems1–2. Murayama and Okada3 showed that Sendai virus-induced fusion of Ehrlich ascites cells required Ca2+. When Ehrlich ascites cells were treated with Sendai virus in the presence of EDTA, cells agglutinated and lysed but did not fuse3,4. Similarly, Ca2+ was required for the fusion of erythrocytes stimulated by chemicals such as lysolecithin5 or glyceryl monooleate6. Ca2+ plus the ionosphere A23187 (ref. 7) or Ca2+ at high pH (ref. 8) promoted the fusion of chicken erythrocytes. Fusion of phosphatidylserine-rich liposomes was absolutely dependent on the presence of Ca2+9. We describe here the fusion of human erythrocyte ghosts promoted by the combined action of Ca2+ and phosphate buffer. Human erythrocyte ghosts, because they lack cytoplasm and are easily filled with small or large molecules10,11 are useful for the investigation of membrane fusion.

The non-parallel increase of amylase, chymotrypsinogen and procarboxypeptidase in the developing chick pancreas

Abstract Developmental changes in the levels of α-amylase, procarboxypeptidase and chymotrypsinogen were studied in the pancreas of the chick embryo (developmental age 13–20 days) and the newly-hatched chick (development age 21–23 days). 1. 1.|Levels of zymogens were determined by measuring the activity of the corresponding proteolytic enzyme after activation of the zymogen with trypsin. For this purpose controlled conditions of activation of procarboxypeptidase and chymotrypsinogen were developed which would permit full activation of the zymogens in homogenates of pancreas at different stages of development. 2. 2.|When homogenates of pancreas at different stages of development were mixed, enzyme activities were additive. This seems to rule out the possibility that changing concentrations of inhibitors or activators during development influence the activities of the enzymes studied. 3. 3.|The specific activities of all three enzymes increase most steeply after 18 days of development reaching a maximum in the 1-day chick (22 days of development). However, in spite of this similarity, the level of each enzyme changes with age in a different and characteristic manner. From 13–22 days of development the specific activities of amylase, carboxypeptidase and chymotrypsin increase 9-fold, 24-fold and 200-fold, respectively.

A quantitative study of ultramicroinjection of macromolecules into animal cells

Improvements in the technique of ultramicroinjection of macromolecules into animal cells are described. The method is based on the Sendai virus-induced fusion of animal cells with erythrocyte ghosts containing trapped macromolecules. Fusion of hepatoma tissue culture (HTC) cells with ghosts prepared by hemolysis of erythrocytes in the presence of cytochrome C is much more efficient than fusion with ghosts prepared in the presence of bovine serum albumin (BSA) as in previous investigations. La+++ is more fficient in promoting fusion and less toxic to cells than Mn++, which was used previously. Thus in all subsequent experiments, erythrocytes were hemolyzed in the presence of cytochrome C plus other macromolecules to be trapped, and the resultant ghosts fused in the presence of La+++. The percentage of HTC cells which fused with ghosts reached 80% in many experiments. Ghosts containing 125I-BSA were used to measure the number of BSA molecules injected into HTC cells. About 10(6) BSA molecules were injected per fused cell. The overall efficiency of injection was low (about 0.02% of the starting material).

Glutamine-stimulated modification and degradation of glutamine synthetase in hepatoma tissue culture cells

Effects of glutamine on glutamine synthetase (GS) activity of hepatoma tissue culture (HTC) cells were studied with the aid of a specific goat anti-rat GS serum. Immunodiffusion and immunoelectrophoretic tests show that rat liver GS and HTC cell GS are immunologically similar but not identical. Immunotitrations of HTC cell extracts demonstrate that in cells incubated in high concentrations (5 mM) of glutamine, a cross-reacting form of GS with a decreased enzyme-specific activity accumulates. On prolonged incubation of cells in high glutamine, there is net degradation of GS to form immunologically inactive products. Radio-immunoprecipitation experiments show that glutamine acts by accelerating the degradation of preformed GS.

The effect of hydrocortisone on the accumulation of amylase in embryonic chick pancreas

During the late stages of development of the chick embryo amylase and other exportable enzymes accumulate rapidly in the pancreas from about 17 days of development until 2 days after hatching (develop mental age 22 days) [l-3] . Although this phase of rapid accumulation of digestive enzymes is dramatic and apparently plays a vital role in pancreatic development, it is clearly the consequence of the maturation of an already highly differentiated organ [4]. immediately prior to the maturation phase (from 12 to 16 days of development) amylase specific activity remains constant. These observations implied that there must be an early phase of differentiation before 12 days of development, which should be characterized by the rapid accumulation of amylase. The present study shows that there is indeed an early phase of differentiation which starts before 6 days of develop ment and that during this phase, which ends at 12 days of development, the specific activity of amylase increases by more than 2 orders of magnitude. Pancreases from 7-day embryos cultured in vitro underwent a limited degree of differentiation as indicated by the accumulation of amylase. The rate of accumulation of amylase in pancreas cultured in vitro could be greatly increased by adding hydrocortisone to the medium even after accumulation in the absence of the hormone had stopped.

The effect of hydrocortisone on exportable enzyme accumulation in the differentiating chick pancreas

Abstract Developmental patterns of accumulation of chymotrypsinogen, procarboxypeptidase A, amylase, and endonuclease in the embryonic chick pancreas were compared. The enzymes studied seem to fall into two classes: (1) those, like amylase and procarboxypeptidase, which start to accumulate rapidly at early stages of development and which accumulate in two steps; and (2) those, like chymotrypsinogen and endonuclease, which accumulate slowly during early development (before 14 days) and rapidly thereafter in a single step. Hydrocortisone (hemisuccinate) injection at 10 and 12 days of development causes a precocious increase in the levels of amylase, procarboxypeptidase A, chymotrypsinogen, and, to a lesser extent, endonuclease. The levels of chymotrypsinogen and endonuclease increase during the first day after hydrocortisone injection, whereas the levels of amylase and procarboxypeptidase A are elevated only after a lag of at least a day. Effects of hydrocortisone injection on acinar cell ultrastructure were also investigated. Injection of the hormone prematurely induced a rapid proliferation of the rough endoplasmic reticulum and a sharp increase in the number and size of zymogen granules. The data support the hypothesis that the exocrine chick pancreas differentiates in two phases, an early phase and a maturation phase and that the latter phase is initiated by adrenal cortical steroids secreted by the embryo.

Patterns of growth and α-amylase activity in the developing chick pancreas

Abstract 1. 1. Developmental changes in total pancreatic protein, DNA, RNA and α-amylase (α-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) activity were studied in chick embryos aged 10–20 days and in 1–5-day-old chicks (developmental age 21–25 days). 2. 2. The protein content of the pancreas increases rapidly from 10 to 13 days, more slowly from 13 to 18 days, very rapidly from 19 to 22 days and again more slowly from 22 to 25 days. 3. 3. A virtually constant concentration of RNA per mg of protein is observed from 13 to 25 days of development. On the other hand, the concentration of DNA per mg of protein, which is constant from 13 to 18 days, falls sharply between 18 and 21 days. 4. 4. The specific activity of amylase remains constant from 11 to 16 days of development and increases about 8-fold from 16 to 22 days, after which it falls. At its maximum concentration (22–23 days) amylase constitutes about 12% of the total pancreatic protein. 5. 5. It seems significant that the steepest rise in amylase activity between 18 and 22 days coincides with the period of rapid growth and increase in cell size which overlaps the time of hatching.

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.