Cell Biochemistry and Biophysics

, Volume 61, Issue 3, pp 619–628

Q2N and S65D Substitutions of Ubiquitin Unravel Functional Significance of the Invariant Residues Gln2 and Ser65

  • Pradeep Mishra
  • C. Ratna Prabha
  • Ch. Mohan Rao
  • Srinivas Volety
Original Paper

DOI: 10.1007/s12013-011-9247-8

Cite this article as:
Mishra, P., Ratna Prabha, C., Rao, C.M. et al. Cell Biochem Biophys (2011) 61: 619. doi:10.1007/s12013-011-9247-8

Abstract

Ubiquitin is a small, globular protein, structure of which has been perfected and conserved through evolution to manage diverse functions in the macromolecular metabolism of eukaryotic cells. Several non-homologous proteins interact with ubiquitin through entirely different motifs. Though the roles of lysines in the multifaceted functions of ubiquitin are well documented, very little is known about the contribution of other residues. In the present study, the importance of two invariant residues, Gln2 and Ser65, have been examined by substituting them with Asn and Asp, respectively, generating single residue variants of ubiquitin UbQ2N and UbS65D. Gln2 and Ser65 form part of parallel G1 β-bulge adjacent to Lys63, a residue involved in DNA repair, cell-cycle regulated protein synthesis and imparting resistance to protein synthesis inhibitors. The secondary structure of variants is similar to that of UbF45W, a structural homologue of wild-type ubiquitin (UbWt). However, there are certain functional differences observed in terms of resistance to cycloheximide, while there are no major differences pertaining to growth under normal conditions, adherence to N-end rule and survival under heat stress. Further, expression of UbQ2N impedes protein degradation by ubiquitin fusion degradation (UFD) pathway. Such differential responses with respect to functions of ubiquitin produced by mutations may be due to interference in the interactions of ubiquitin with selected partner proteins, hint at biomedical implications.

Keywords

Ubiquitin Ubiquitin structure Ubiquitin function G1 β bulge of ubiquitin Mutations of ubiquitin Structure–function relations in ubiquitin 

Introduction

Functional morphology of a protein is entirely determined by its sequence. Smaller proteins with vital functions are known to have longer unit evolutionary periods and permit very few substitutions in their amino acid sequence. Ubiquitin, a small protein of 76 amino acid residues and ubiquitous distribution, plays myriad roles in eukaryotic cells. It is known to be involved in cell-cycle regulation [1] DNA packing [2], DNA repair [3], transcriptional regulation [4], degradation of proteins by proteasome [5, 6] and lysosome [7]. Ubiquitin is attached post-translationally through its C-terminal to the side chain of Lys on a target protein forming an isopeptide linkage [8]. This post-translational modification is referred to as ‘ubiquitination’. Ubiquitination can be of three types [9], namely, monoubiquitination, polyubiquitination and multiubiquitination. The site of ubiquitination on the substrate, the length of ubiquitin chain and the lysine of ubiquitin involved in the linkage in a polyubiquitin chain combined together determine the destiny of the substrate protein, either to serve a specific function or to undergo degradation. Defective or excessive ubiquitination of several proteins is the molecular basis for many cancers, neurodegenerative diseases and hereditary disorders [10, 11]. By virtue of the multitudinous roles ubiquitin plays, it interacts with several proteins and sometimes more than one protein simultaneously, making its structure and topology extremely important for ubiquitin–protein interactions. Smaller size and near total conservation of its sequence among all eukaryotes underline the fact that nature has perfected structure–function relations in ubiquitin early on in eukaryotic evolution [12, 13, 14, 15, 16, 17]. Sequence comparisons of yeast, human and plant ubiquitins revealed 73 residues remain invariant out of a sequence of 76 residues, indicating their importance in structure and function of the protein. In the present study we addressed the functional significance of two invariant residues, Ser65 and Gln2, by substituting them with Asp and Asn, respectively. These two residues are found in the same position in homologues and show greater preference for adopting same kind of secondary structure [18]. Our results show that there is only marginal change in the structure of the molecule. However, these mutations affect numerous functions of ubiquitin to different degrees, establishing that these residues are important for some of the functions, while others remain unaffected by topological changes in this region of the molecule.

Ubiquitin is a globular protein lacking disulphides, cofactors and other post-translational modifications. The structure of ubiquitin has a hydrophobic core hidden in an ensemble of five strands of β-sheet and an α-helix assembled with nine reverse turns [19]. The resultant structure of the molecule is extensively hydrogen bonded, compact and highly heat stable. Folding studies on ubiquitin using pulsed H–D exchange NMR experiments indicated that the N-terminal β-sheet and α-helix are folded early, while the C-terminal half of the protein displayed slow folding kinetics [20].

The role of individual residues in the structure or function of ubiquitin cannot be comprehended by sequence comparisons among different species due to the absence of sequence diversity in ubiquitin. However, site-directed mutagenesis has been employed in several cases to understand specific functions of many residues [21, 22, 23, 24]. In order to include an effective intrinsic fluorophore in the sequence of ubiquitin, UbF45W was constructed by substituting Phe45 with Trp. The folding kinetics and stability were studied using fluorescence, and the protein was found to behave like wild-type protein [25].

The first β bulge of ubiquitin located in the N-terminal portion of the protein has several unusual features [19] and it folds early on in the refolding experiments. The importance of first β bulge in the structure of ubiquitin has been the subject of many investigations [24, 26, 27].

The second β-bulge of ubiquitin, formed by Glu64 (1), Ser65 (2) and Gln2 (X) (Fig. 1) is adjacent to Lys63. Lys63 has been found to be important for UV repair of DNA [28] and resistance of cells to stress conditions such as exposure to protein synthesis inhibitors [28, 29] and endosomal degradation of certain proteins [7]. Some remarkable features displayed by the second β-bulge are firstly, it is a parallel G1 β-bulge, a rare feature in proteins, and secondly, Glu64 is the first residue in the β-bulge, a position generally occupied by Gly [19]. The relevance of Glu64 in the structure and function of ubiquitin was investigated earlier by generating a variant of ubiquitin UbE64G, in which Glu64 was replaced by Gly [24]. Studies on percent preference of occurrence of various amino acids in G1 β-bulge revealed that the amino acids Ser and Gln have lower preference for the second and X positions of β-bulge [30]. In spite of their low propensity for β-bulge, the residues Ser65 and Gln2 are found to be universally conserved in the sequence of ubiquitin.
Fig. 1

The position of parallel β-bulge in the structure of Ubiquitin

In the present study, an attempt has been made to unravel the importance of Ser65 and Gln2 of the parallel G1 β-bulge in the structure, stability and function of ubiquitin. To attain the objective, the residues Ser65 and Gln2 of parallel G1 β-bulge have been replaced by Asp and Glu, respectively, to generate two single mutants of ubiquitin UbS65D and UbQ2N. The residue substitutions have been chosen by comparing the sequence of ubiquitin with its structural homologues SMT3, SUMO1, SUMO2, SUMO3, NEDD8 and RUB1 (Table 1) [18]. Besides, the replacing residues show greater preference for the given positions in G1 β-bulge. The percentage frequencies of occurrence of Ser65 and Gln2 in β-bulge are 1.5 and 0.34, respectively, which are much lower than those of Asp and Asn at 2.4 and 5.4, respectively [30].
Table 1

The sequences of SMT3, SUMO1, SUMO2, SUMO3, NEDD8 and RUB1 have been aligned with that of ubiquitin to select the residues for replacement Ser65 and Gln2 of β-bulge (indicated in bold) [29]

 

1

2

3

4

5

6

7

8

9

10

60

61

62

63

64

65

66

67

68

69

70

UBWt

M

Q

I

F

V

K

T

L

T

G

N

I

Q

K

E

S

T

L

H

L

V

SMT3

I

N

L

K

V

S

D

G

S

D

M

E

D

N

D

I

I

E

A

H

SUMO1

I

K

L

K

V

I

G

Q

D

S

G

M

E

E

E

D

V

I

E

V

Y

SUMO2

I

N

L

K

V

A

G

Q

D

G

E

M

E

D

E

D

T

I

D

V

F

SUMO3

I

N

L

K

V

A

G

Q

D

G

R

M

E

D

E

D

T

I

D

V

F

NEDD8

M

L

I

K

V

K

T

L

T

G

K

I

L

G

G

S

V

L

H

L

V

RUB1

M

I

V

K

V

K

T

L

T

G

H

L

V

E

G

M

Q

L

H

L

V

The variants of ubiquitin UbS65D and UbQ2N engineered by site-directed mutagenesis have been characterized by circular dichroism (CD) and fluorescence spectroscopy. Our results show that the mutant proteins are structurally similar to wild-type protein (UbWt).

The genes carrying mutations for UbS65D and UbQ2N were expressed in polyubiquitin gene UBI4 deletion mutant of S. cerevisiae, to assess their functionality by complementation. Growth kinetics, adherence to N-end rule and degradation by ubiquitin fusion degradation (UFD) pathway were monitored initially under stress-free conditions to probe whether there would be any detrimental effects produced in the organism due to the mere presence of UbS65D and UbQ2N [31, 32, 33]. The mutant proteins UbS65D and UbQ2N were tested for their ability to functionally complement and rescue UBI4 mutants of S. cerevisiae under various stress conditions, since the polyubiquitin gene UBI4 is expressed under stress and rescues the organism under extreme conditions [34]. Though the mutant proteins are structurally very similar to the control protein UbF45W, they show several functional deficiencies, implicating roles for the conserved residues in the functional integrity of the molecule.

Materials and Methods

SUB60 (MATa ubi4-_2::LEU2 lys2-801leu2-3,112 ura3-52 his3-D200 trp1-1) and SUB62 (MATa lys2-801 leu2-3,112 ura3-52 his3-D200 trp1-1) strains of S. cerevisiae [34] were used for in vivo studies.

Construction of UbS65D and UbQ2N Plasmids Using Site-Directed Mutagenesis

In the present study, the wild-type ubiquitin (UbWt) and the fluorescent variant of ubiquitin (UbF45W) were used as controls. The two variants UbS65D and UbQ2N were generated from UbF45W to facilitate near-UV-CD and fluorescence spectroscopic investigations.

Ubiquitin gene with F45W mutation [25] from pKK223-3 was subcloned into pUC18. The single mutations for producing UbS65D and UbQ2N were introduced following Genei in vitro site-directed mutagenesis protocol. Incorporation of the mutations was confirmed by sequencing. The mutated fragments from pUC18 vector were cloned back in the original vector, and pKK223-3-S65D and pKK223-3-Q2N were obtained.

Purification of UbWt and Its Variants UbF45W, UbS65D and UbQ2N

The UbWt and its variants UbF45W with single residue substitution, and the double mutants with UbS65D and UbQ2N carrying F45W mutation were purified using the methods reported in the literature [22, 35, 36, 37, 38, 39, 40]. Since UbWt is a thermostable protein, heat treatment was employed as one of purification steps, and the same was followed during the purification of UbF45W [25], UbE64G [24], UbS65D and UbQ2N as well. In addition, the changes introduced in the protocol for purification of UbE64G were adapted for purification of UbS65D and UbQ2N [24]. The protein stocks were stored in 10% glycerol at −20°C.

CD and Fluorescence Spectroscopy of UbF45W, UbS65D and UbQ2N

CD spectra of UbWt, UbF45W, UbS65D and UbQ2N were recorded using a Jasco J-715 spectropolarimeter. Far-UV-CD spectra were recorded using 1-mm path length cells in the wavelength range of 200–250 nm with a scan speed of 50 nm/s. The spectra were accumulated for five times to improve the signal-to-noise ratio. Protein solutions were prepared in 10 mM Tris–HCl, pH 7.4 and concentration of protein was 0.2 mg/ml (23.4 μM). The near-UV CD spectra were recorded using 1 cm path length cells in the wavelength range of 250–320 nm. Protein concentration was 1 mg/ml (117 μM). All spectra were corrected with appropriate blanks.

Fluorescence spectra of the samples were recorded using Hitachi F-4010 fluorescence spectrophotometer with excitation and emission band-passes of 5 nm. Protein solutions were prepared in Tris–HCl buffer, pH 7.4 with protein concentration of 23.4 μM (0.2 mg/ml). Samples were excited at 280 nm, and the intrinsic fluorescence of tryptophan was recorded in the range of 300–400 nm.

Construction of Yeast Vectors Carrying UbS65D and UbQ2N Variants of Ubiquitin Gene

The yeast–bacteria shuttle vector YEp96 [28] was a generous gift from Prof. Daniel Finley. The vectors YEp96/F45W, YEp96/S65D and YEp96/Q2N were constructed by replacing Ub gene in YEp96 present under CUP-1 promoter with UbF45W, UbS65D and UbQ2N variants of the gene, respectively. Expression of the genes can be induced in yeast by 10–100 μ M copper sulphate. The plasmids YEp96/S65D and YEp96/Q2N were sequenced for confirmation.

Effect of Mutations on the Growth of Yeast Cells

The cultures of SUB60 cells, transformed by plasmid YEp96/Wt carrying wild-type ubiquitin gene, YEp96/F45W, YEp96/S65D and YEp96/Q2N plasmids with mutated variants of ubiquitin gene, were grown at 30°C at 200 rpm, except where indicated in synthetic dextrose medium consisting of 0.67% Hi-media yeast nitrogen base supplemented with uracil, leucine, tryptophan, lysine, histidine and 2% glucose as carbon source, as and when required. 100 μM copper sulphate was used as an inducer to check the effects of expression of mutant ubiquitin genes on S. cerevisiae cells. Optical density at 600 nm was measured every 2 h. Their growth was compared with wild-type strain for UBI4, SUB62 [34].

Effect of the Ubiquitin Mutation on Substrate Protein Turnover by Adherence to N-End Rule and by UFD Pathway

S. cerevisiae strains SUB60 and SUB62 were transformed by pUb23. The transformants of SUB60 by plasmids YEp96/Wt, YEp96/F45W, YEp96/S65D and YEp96/Q2N were also transformed by pUB23. The plasmid pUb23 is a 2 μ-based vector expressing ubiquitin-X-βGal (Ub-X-βGal) fusion gene under the control of galactose-inducible GAL10 promoter with X position as Met and Pro in independent sets to test the effects of mutations on the degradation by adherence to N-end rule [41, 42] and by UFD pathways [41, 43], respectively. pUb23 has URA as selection marker. The transformants were grown to mid-log phase in synthetic galactose media at 30°C, conditions under which Ub-X-βGal is constitutively expressed. The cells were grown in two independent sets. In the first set, 100 μM of CuSO4 was added for the induction of UbWt, UbF45W, UbS65D and UbQ2N from YEp96 and in the second set, consisting of controls CuSO4 was not added. β-galactosidase activity was assayed to measure protein stability. The enzyme assays were repeated thrice in independent sets and the mean values have been presented with error bars.

Complementation Assay

As mentioned in the "Introduction" section, the polyubiquitin gene UBI4 present in yeast expresses under stress conditions and is primarily responsible for the survival of the organism [34]. Stress-hypersensitive UBI4 mutant, the SUB60 strain of yeast was transformed by YEp96 plasmid carrying mutated ubiquitin gene under CUP1 promoter and was tested for complementation under stress. SUB60 yeast cells complemented by UbWt expressed from the plasmid, and thus rescued from stress were used as positive control.

Heat Sensitive Test

The vector-carrying variant of ubiquitin was transformed into yeast UBI4 deletion mutant SUB60. The UBI4 deletion mutant can grow in the absence of the gene at 30°C (permissive temperature), but fails to grow at 39°C. Complementation by functional ubiquitin expressed from a plasmid can rescue the cells. Heat sensitivity test was performed to confirm the functional integrity of the mutant ubiquitins [32]. The transformants of SUB60 by plasmids YEp96/Wt, YEp96/F45W, YEp96/S65D and YEp96/Q2N were grown to log phase till the optical density of the cultures reaches a value between 0.5 and 0.6. Fourfold serial dilution of these cells was made and were plated on SD selection media with and without induction by 100 μM copper sulphate. Plates were incubated at 40°C for variable time periods of 0, 4, 8, 12 and 16 h, shifted back to 30°C, and the colonies were counted. The experiment was repeated three times in independent sets and the mean values have been presented with error bars.

Antibiotic Sensitivity Test

Complementation potential of the ubiquitin variants, UbS65D and UbQ2N, was investigated using antibiotic sensitivity test [44]. SUB60 transformants with plasmids YEp96/Wt, YEp96/F45W, YEp96/S65D and YEp96/Q2N were grown to log phase with their optical density values around 0.2 and serially diluted three fold and spotted on YPD media with 100 μM copper sulphate and cycloheximide (4 μg/ml). Plates were incubated for 10 days for assessment of revival and growth.

Results

CD and Fluorescence Studies of the Variants of Ubiquitin UbF45W, UbS65D and UbQ2N

Far-UV CD spectra of the wild type UbF45W and its variants UbS65D and UbQ2N are shown in Fig. 2a. The far-UV CD spectrum of UbQ2N is almost identical to that of UbF45W. However, the spectrum of UbS65D shows marginal changes in secondary structure content, compared with the control UbF45W. The CD spectrum of a protein in the near-UV spectral region (250–320 nm) offers information on the tertiary structure of proteins, which is generally a qualitative measure of the overall conformation of proteins [45, 46, 47]. Based on near-UV CD spectra, the tertiary structures of UbS65D and UbQ2N show minor differences with respect to UbF45W, indicating that the tertiary structure of the protein remains mostly unaffected by the single residue substitutions (Fig. 2b). Thus, the results obtained from CD studies indicate no significant alterations in the structural features of the proteins.
Fig. 2

a Far-UV CD spectra of three forms of ubiquitin, UbF45W (1), UbS65D (2) and UbQ2N (3). b Near-UV CD spectra three forms of ubiquitin, UbF45W (1), UbS65D (2) and UbQ2N (3)

We further studied the stability of the proteins by fluorescence spectroscopy. Guanidinium chloride-induced denaturation profiles of UbF45W and UbS65D were almost coincident with identical C1/2 values (Fig. 3). The C1/2 value of guanidinium chloride (GdmCl) for UbQ2N is 0.35M lower than that of UbF45W (Fig. 3). These results indicate that stability of the protein UbS65D is not compromised as a consequence of the mutation, whereas UbQ2N has become mildly unstable compared to the wild type.
Fig. 3

Gaunidinium chloride denaturation curves of UbF45W (1), UbS65D (2) and UbQ2N (3). The denaturation of the proteins was followed by recording the intrinsic fluorescence spectra of Trp by exciting it at 280 nm. The wavelength of maximum emission was plotted against concentration of guanidinium chloride

Functional Complementation Studies with UbQ2N and Ubs65d

Ubiquitin is encoded by four different genes in S. cerevisiae. Among them UBI1, UBI2 and UBI3 are constitutive, while UBI4 is expressed to ensure survival under various stress conditions. UBI4 gene cluster expresses 4–5 copies of ubiquitin as a single polypeptide chain, which is processed into ubiquitin molecules post-translationally. SUB60 mutants lacking UBI4, which though fail to withstand stress conditions, grow normally at 30°C. SUB62 cells are wild type for UBI4. In the following experiments, SUB60 cells were transformed with yeast plasmids (carrying variants of ubiquitin gene as indicated), YEp96/UbWt, YEp96/UbF45W, YEp96/UbE64G, YEp96/UbS65D and YEp96/UbQ2N. The above plasmids carrying ubiquitin gene under CUP-1 promoter, have tryptophan auxotrophy complementation marker for selection. SUB62 and SUB60 were used as positive and negative controls, respectively. SUB60 cells transformed with YEp96/UbWt and expressing UbWt behave as SUB62 cells do.

Effects of UbQ2N and UbS65D Expression on Growth of S. cerevisiae

Ubiquitin is known to participate in a number of physiological processes in the cell. Hence, if the mutant forms of ubiquitin are non-functional, they may interfere with and slow down the cellular phenomena by mixing with the cellular pool of ubiquitin and acting as competitive inhibitors for ubiquitin interacting proteins and enzymes. As a consequence they may affect the growth rate and survival of the organism. To test whether the mutant proteins UbS65D and UbQ2N have any such influence over the organism, the mutated genes under copper sulphate-inducible CUP1 promoter were expressed in yeast cells. SUB62 strain and SUB60 strains transformed by UbWt and UbF45W were used as controls for growth conditions. However, these mutants do not show significant differences in growth and survival of the latter organisms when compared to UbWt and UbF45W controls under normal conditions (Fig. 4). Hence, under normal conditions, the presence of the variants UbS65D and UbQ2N were not detrimental to growth of the organism; in other words, they do not behave as non-functional competitive inhibitors, which can poison the cellular pool of ubiquitin.
Fig. 4

Growth curves of UBI4 mutants of SUB60 cells of S. cerevisiae transformed by YEp96/Wt, YEp96/S65D and YEp/Q2N expressing UbWt, UbS65D and UbQ2N, respectively. SUB62 strain, which is wild type for UBI4 and untransformed SUB60 strain were used as positive and negative controls, respectively

Degradation of Protein Substrates by N-End Rule and UFD Pathways

Translational fusions of ubiquitin with β-galactosidase were constructed earlier to study the effect of various N-terminal residues on the turn-over of β-galactosidase. In the construct Ub-X-β-gal, X could be any one among the twenty amino acids. The longevity of the protein β-galactosidase was determined based on whether X was a stabilizing or destabilizing residue, which is termed as the N-end rule [41, 42]. Ub-X-β-gal was found to be deubiquitinated in all cases, except with Ub-Pro- β-gal, where X was Pro. Further, the N-terminal ubiquitin acted as a degradation signal leading to degradation of β-galactosidase by UFD pathway [41, 43]. In order to measure the protein stability, β-galactosidase activity was assayed in the double transformants of SUB60 cells carrying pUB23 along with YEp96/UbWt, YEp96/UbF45W, YEp96/UbS65D and YEp96/UbQ2N. Two fusions of ubiquitin with β-galactosidase, namely, Ub-Met-β-gal and Ub-Pro-β-gal were selected to study the effect of mutations on adherence to N-end rule and UFD pathways, respectively. β-galactosidase activity remained more or less unchanged in YEp96/Wt transformants expressing Ub-Met-β-gal. The translational fusion of Ub-Met-β-gal undergoes deubiquitination and hence, has longer half-life as Met is a stabilizing residue. It did not show any change in degradation rate with the presence of mutant ubiquitin molecules in the cell (Fig. 5a). However, β-galactosidase activity showed a decline, when Pro was present as the N-end residue in YEp96/Wt transformants. Ub-Pro-β-gal which is a short-lived fusion under normal conditions, as it undergoes polyubiquitination on the Lys48 residue of N-terminally fused ubiquitin. Conversely, Ub-Pro-β-gal showed prolonged half-life in the presence of mutant ubiquitin UbQ2N (Fig. 5b). UbS65D did not seem to have any effect over degradation by UFD pathway. An earlier finding relevant to the above observation is that β-galactosidase in Ub-X-β-gal with Met or any amino acid residues other than Pro in the position of X loses the N-terminal ubiquitin with the help of deubiquitinating enzymes. The β-gal portion of the fusion protein is later ubiquitinated by N-recognin and degraded. On the other hand, Ub-Pro-β-gal undergoes ubiquitination by means of ubiquitin-conjugating enzymes, UBC4 and UBC5, and ligations to ubiquitin by UFD2 and UFD4. The difference observed in the degradation rate could be due to decreased efficiency of UFD pathway of degradation arising out of encountering UbQ2N.
Fig. 5

Effect of UbS65D and UbQ2N on the half-lifves of proteins in UBI4 background. S. cerevisiae strains SUB60, SUB62 and SUB60 transformed by plasmids YEp96/Wt, YEp96/F45W, YEp96/S65D and YEp/Q2N expressing the variant forms of ubiquitin, namely UbWt, UbF45W, UbS65D and UbQ2N, respectively. These cells were also transformed by pUB23 expressing a Ub-Met-β-galactosidase fusion, a substrate which undergoes deubiquitination and subsequently degradation by N-end rule pathway, b Ub-Pro-β-galactosidase, a substrate which undergoes degradation by UFD pathway. Met and Pro are the N-terminal residues of β-galactosidase fusion. SUB60 and SUB62 strains were used as negative and positive controls, respectively

Complementarity of Q2N and S65D in UBI4 Mutants under Heat Stress

The mutations did not have any negative effect with respect to heat stress. During heat stress, the misfolded and unfolded proteins accumulate, imposing a stress on the ubiquitin proteasome system. In wild-type cells, UBI4 is induced to meet the increased demand for ubiquitination. In SUB60 mutant where the gene is absent, the cells fail to degrade the misfolded proteins because of paucity of ubiquitin and become lethal. In this study, SUB60 cells expressing UbS65D and UbQ2N have shown same levels of survival as SUB62 cells, which have ubiquitin expressed from CUP1 promoter of the plasmid (Fig. 6). Hence, the mutations do not seem to affect the conjugation of ubiquitin and polyubiquitin chain formation. Therefore, ubiquitinating enzymes acting on unfolded and misfolded proteins do not apparently discriminate UbS65D and UbQ2N molecules from UbWt. Preliminary evidences from our laboratory, arising out of experiments studying incorporation of UbS65D and UbQ2N in polyubiquitin chain formation using western blot analysis, support the above conjecture (Pradeep Mishra and C. Ratna Prabha, unpublished observations).
Fig. 6

Functional complementation under heat stress by the variants of ubiquitin UbS65D and UbQ2N in UBI4 deletion mutant strain SUB60 of S. cerevisiae. SUB60 cells were transformed by plasmids YEp96/UbWt, YEp96/F45W, YEp96/S65D and YEp/Q2N. SUB62 and SUB60 were positive and negative controls, respectively

Complementation of Q2N and S65D in UBI4 Mutants under Cycloheximide Stress

The antibiotic stress complementation analysis with mutant forms of ubiquitin shows that the mutations have drastic effect on survival of SUB60 cells upon exposure to cycloheximide (Fig. 7). The β-bulge of ubiquitin seems to play a crucial role in overcoming the antibiotic stress. Alterations in ubiquitin structure do not allow the variants to play complementary role.
Fig. 7

Antibiotic stress complementation of Saccharomyces cerevisiae SUB60 strain by variants of ubiquitin UbQ2N and UbS65D. The strains SUB62, SUB60 were the positive and negative controls, respectively. SUB60 cells transformed by YEp96/Wt, YEp96/F45W, YEp96/E64G, YEp96/S65D and YEp96/Q2N expressing UbWt, UbF45W, UbE64G, UbS65D and UbQ2N, respectively, in the UBI4 deletion were tested for cycloheximide resistance. Undiluted stock and threefold serial dilutions (2), (3) and (4) spotted on YPD plates in two sets. First set contained no cycloheximide and second set contained 4 μg/ml of cycloheximide. 100 μM copper sulphate was used as inducer. The SUB60 and SUB60 strains transformed by UbE64G, UbS65D and UbQ2N fail to grow in the presence of cycloheximide

Discussion

The role of ubiquitin is pivotal to cell physiology. Many structurally and functionally diverse types of proteins which interact with ubiquitin are firstly, the enzymes of ubiquitination pathway; secondly, the recipient proteins often referred to as the substrates; and finally the deubiquitinating enzymes [8, 48, 49, 50]. All of these proteins establish direct physical contact with ubiquitin through covalent and non-covalent interactions. Interestingly, all seven lysines of ubiquitin are conjugated to different recipients or substrate proteins and designate them to serve entirely different purposes [51, 52, 53, 54, 55]. It is also known that unrelated motifs belonging to at least eight different groups of proteins interact with ubiquitin. Hence, it is tempting to assume that different facets of ubiquitin form the interactive surfaces in diverse cases. In the present study, we have addressed the structural and functional roles of Ser65 and Gln2 in the C-terminal β-bulge of ubiquitin by creating variants UbS65D and UbQ2N. Substitution of conserved residues Ser65 and Gln2 by Asp and Asn, respectively, in the wild-type protein does not affect their secondary structure, introducing minor and subtle changes in the tertiary structure as monitored by far-UV CD spectroscopy. However, intrinsic fluorescence spectra show that the environment around Trp residue has changed in both the variants and the change is greater in UbS65D than in UbQ2N.

Functionally, ubiquitin is well known to interact with other proteins, while accomplishing its myriad roles. Protein–protein interactions impose evolutionary constraint of maintaining their topological features on interactive proteins. This evolutionary compulsion leaves two choices with the interactive partners, either to undergo co-evolution in a concerted fashion or to maintain the structure–function relation by conserving their primary structure and shunning evolutionary changes, especially of the interactive surface. Smaller proteins like ubiquitin with multiple interactive partners apparently choose the second alternative. In the present study, parallel β-bulge with residues unusual for their location, continues to maintain them because of functional reasons. Though only three residues in ubiquitin have been shown to interact with other proteins, the residues of parallel β-bulge seem to influence several functions of the molecule most likely by altering the overall topology of the molecule.

For functional integrity analysis, the proteins were expressed in host cells lacking UBI4 gene product [34]. Since the protein UBI4 is not required for normal growth, the cells remained unaffected. Besides, the variants do not seem to contaminate the cellular pools of ubiquitin, as their expression did not have any detrimental effect on the organism. Similar behaviour is observed with UbK63R, where Lys63 of ubiquitin is replaced by Arg [28, 31, 44, 52].

Under heat stress, the UBI4 mutants face scarcity of ubiquitin to deal with the accumulating bulk of misfolded and unfolded proteins. Failure of degradation of the non-functional aggregates results in lethality. However, expression of the variants in UBI4 background results in survival of the organism, indicating their ability to replace the UbWt in polyubiquitin chain formation. These mutations are located in the neighbourhood of Lys63 that plays a crucial role in the polyubiquitin chain formation and UPS-mediated degradation of proteins [29, 32, 53].

Ubiquitin influences cellular protein homeostasis in two ways, at both the levels of protein synthesis and protein degradation. Protein L28 present in the large subunit of ribosome has been found to be poly-ubiquitinated [31]. This highly conserved modification of the protein found even in humans, is regulated by cell cycle. In this case, ubiquitin in the polyubiquitin chain forms isopeptide linkages through its Lys63. In UBI4 mutants, mutation of Lys63 to Arg in UbK63R, interfered with the modification, rendering the cells more sensitive to protein synthesis inhibitors, for instance, cycloheximide. The substitution mutation of residues 64 and 65 belonging to β-bulge, being in the immediate neighbourhood of Lys63 and that of Gln2 due to its proximity to Lys63 by virtue of tertiary structure, appear to impair some of ubiquitin’s functions involving Lys63, such as resistance to protein synthesis inhibitors. In addition, treatment with cycloheximide often leads to one more effect of increasing the load of truncated peptides for ubiquitin-mediated proteasomal degradation, and creating a scarcity of ubiquitin may be contributing to the lethality observed with the mutant. However, the results from thermal tolerance experiments suggest that UbS65D and UbQ2N can supplement ubiquitin pool for polyubiquitin chain formation on unfolded and misfolded proteins. Hence, the reason for phenotype being observed with either UbQ2N or with UbS65D, resembling UbK63R, can presumably be interpreted as an effect because of failure of ubiquitination of ribosomal protein L-28.

Since a vast variety of proteins interact with ubiquitin, it is important to understand the molecular mechanism underlying this phenomenon, which might eventually be useful in the development of therapeutics. The present study unravels the functional importance of residues in the parallel β-bulge, which are important for degradation of substrates by UFD pathway and also in withstanding the stress induced by cycloheximide.

Malfunctioning of ubiquitin proteasome system is accountable for the pathogenesis of many diseases. Further, ubiquitination of certain important proteins, such as L28, the large ribosomal subunit of yeast, has been shown to be tightly linked to the cell cycle [31]. Rpn4, the regulatory protein for proteasome subunit synthesis [56], and integrases of lentiviruses including that of HIV [57] are well-known N-end rule substrates of UPS. Further, MyoD and latent membrane protein-1 of Epstein-Barr virus have been shown to undergo polyubiquitination in a noncanonical fashion at their N-terminal end [58, 59]. The mutations studied above selectively affect certain functions like degradation of substrates of UFD pathway (fused to ubiquitin through N-terminal) and sensitivity to cycloheximide, indicative of a possibility open for designing strategies for therapeutic intervention of certain implacable maladies, where UPS plays a crucial role.

In conclusion, the present study focuses on the structural and functional importances of residues Gln2 and Ser65 in the parallel β-bulge of ubiquitin. The mutants wherein Gln2 was replaced by Asn and Ser65 was replaced by Asp showed subtle differences in their secondary and tertiary structural features as demonstrated by CD spectral studies. The stability of UbS65D is not altered, whereas that of UbQ2N is marginally lower compared to that of Ub45W. On the other hand, functional analysis established that these mutations appear to be tolerable under normal conditions of growth, but they become detrimental to the survival of the organisms in the presence of protein synthesis inhibitor cycloheximide. Interestingly, they do not affect the survival of the organisms under heat stress. In addition, one of the mutants UbQ2N reduces the degradation rate of UFD pathway substrate Ub-Pro-βgal, where N-terminally fused ubiquitin acts as a degradation signal. The results with translational inhibitor cycloheximide treatment establish that the phenotypes resulting from the mutations in the β-bulge resemble Lys63 mutant, UbK63R. This study demonstrates that mutations in a multifunctional protein, like ubiquitin, need not respond uniformly to all kinds of external cues, and this differential functionality might prove to be useful in designing strategies for therapeutic intervention in certain disease conditions.

Acknowledgments

C.R.P. thanks the University Grants Commission, India, for the research grant. The said author is grateful to Prof. Mark Searle and Prof. Daniel Finley for providing plasmids and strains necessary for the study. The author acknowledges the help received from her students Brinda Panchamia and Mrinal Sharma in the preparation of the manuscript.

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Pradeep Mishra
    • 1
  • C. Ratna Prabha
    • 1
  • Ch. Mohan Rao
    • 2
  • Srinivas Volety
    • 2
  1. 1.Department of Biochemistry, Faculty of ScienceThe Maharaja Sayajirao University of BarodaVadodaraIndia
  2. 2.Centre for Cellular and Molecular BiologyHyderabadIndia

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