Myosin II is important for maintaining regulated secretion and asymmetric localization of chitinase 1 in the budding yeast Saccharomyces cerevisiae

We tested the hypothesis that unusually high chitinase 1 activity previously reported in myo1 strains of the budding yeast Saccharomyces cerevisiae [1] is related to abnormalities in regulated chitinase 1 secretion in vivo. Our results demonstrate that the normally regulated chitinase 1 secretion becomes constitutive and the asymmetric localization of the enzyme in the daughter cell of a mother–daughter pair is altered in a myo1 strain. These results show that myosin II function is important to maintain normal regulation of secretion levels in the cell cycle and the characteristic asymmetric localization of chitinase 1 in yeast cells.

In the budding yeast S. cerevisiae, chitinase 1 is the enzyme responsible for hydrolysis of chitin at the primary septum, resulting in cell separation during cytokinesis. Both the transcription of the CTS1 gene encoding chitinase 1 and the secretion of the enzyme are regulated in a cell cycle-specific manner. The CTS1 gene is cell cycle regulated at the level of transcription in early G1 phase [2,3] and chitinase 1 secretion is temporally regulated, reaching its maximum level following the M/G1 phase transition [4]. While we measure secreted activity in vitro in the external culture medium, chitinase 1 is secreted in a polarized manner in the periplasmic space of the daughter cell where the primary chitin septum is degraded after cytokinesis [5]. It is therefore established that chitinase 1 expression exhibits multiple levels of control. We have previously reported a 20-fold increase in secreted chitinase 1 enzyme activity as a result of myosin II deficiency indicating a possible role for myosin II in regulating its secretion [1].

To test whether regulated secretion is affected in myo1 strains, secreted chitinase 1 activity was measured in supernatants of α-factor-synchronized wild-type (MYO1) and myo1 (myo1Δ∷URA3) strain cultures. In the wild-type strain, no secreted activity is detected during the time interval between 0 and 30 min following release from α-factor-induced G1 arrest (Fig. 1A). The secreted enzyme activity is first detected after 60 min and rapidly reaches the maximum secreted level after 120 min. In our flow cytometry analysis of DNA content, a transition to a negative slope in the DNA content profile, indicated by the arrows in Fig. 1B, represents the approximate time at which the synchronized culture initiates the M/G1 transition. As reflected in the wild-type DNA content profile, the M/G1 phase transition occurs at 120 min (Fig. 1B; see arrow), consistent with the time of maximum secreted enzyme activity for this strain. In the myo1 strain, secreted chitinase 1 activity is already at 50% of the maximum activity at the time interval between zero and 90 min (Fig. 1A). A gradual increase in the secreted enzyme activity begins at 120 min and then reaches the maximum activity at 150 min. As with the wild-type strain, maximum secreted enzyme activity coincides with the 150 min time point identified as the time of M/G1 phase transition in the DNA content profile (Fig. 1B; see arrow). In contrast to the wild-type strain, a higher baseline level of secreted enzyme activity is detected throughout the cell cycle in the myo1 strain, indicative of a shift from regulated to constitutive enzyme secretion. Taken together, these results suggest that myosin II function is necessary to maintain normal regulation of chitinase 1 secretion levels during the cell cycle.

Fig. 1. Dynamics of secreted chitinase 1 enzyme activity.

(A) Secreted chitinase 1 enzyme activity in 1 ml of culture supernatant was assayed as described [4] from wild-type (white columns) and myo1 (gray columns) synchronized cultures at the times indicated following release from α-factor-induced G1 arrest. Enzyme activity is expressed as Fluorescence Units emitted per 4 × 10⁷ cells in a 1-h reaction. Results are averaged from triplicate assays of two experiments with standard deviations shown. (B) Analysis of DNA content by flow cytometry as described [6] was used to assess cell cycle progression in synchronized cultures. The accumulation of cells containing a fully replicated DNA complement (2N) are expressed as the corresponding percentage of the cell population for the wild-type (solid line) and myo1 (dashed line) strains as a function of time in minutes following release from α-factor-induced G1 arrest. The percentages were averaged from three independent experiments with standard deviations shown. Arrows indicate the estimated time of the M/G1 phase transition for each synchronous culture.

The CTS1 gene is expressed during the early G1 phase of the cell cycle [2]. Prior DNA array analysis of cell cycle-regulated genes reported by Spellman et al. [3] indicated that gene transcription data obtained from cells released from α-factor-induced G1 arrest for genes expressed in the M/G1 cluster are not reliable within the first 30 min. We therefore assayed CTS1 mRNA beginning at 30 min through the corresponding G1 phase of the following cell cycle to determine whether transcriptional regulation of CTS1 is altered in the myo1 strain (Fig. 2). In both the wild-type and the myo1 strains, CTS1 gene transcription peaks between 120 and 180 min (Fig. 2). This pattern of expression shows that both strains initiate CTS1 transcription during the M/G1 phase transition described earlier in Fig. 1B. We conclude that the proposed shift to constitutive chitinase 1 secretion is not caused by alterations in CTS1 transcription.

Fig. 2. Semiquantitative RT-PCR assay of CTS1 mRNA.

Total RNA was extracted from cultures of wild-type (diamonds with solid line) and myo1 (squares with dashed line) strains at the times indicated following release from α-factor-induced G1 arrest and 5 µg were used for semiquantitative RT-PCR assays. Sample 2% agarose TBE gels of RT-PCR products amplified from CTS1 mRNA (274 bp) and ACT1 mRNA (608 bp) are presented below the graph. Quantitative analysis was performed by scanning densitometry of the RT-PCR products. The results of four independent experiments were averaged for each time point and the standard error of the mean (SE) was calculated. The ACT1 RT-PCR products [7] from the individual total RNA samples were used as internal RT-PCR controls. To establish the linear amplification range, both RT-PCRs were assayed in independent reactions, varying the number of PCR cycles. The number of cycles selected was determined from the mid-point of the linear regressions shown in the upper right and lower right panels for CTS1 and ACT1, respectively (ordinate, log10 pixel density; abscissa, number of PCR cycles).

Localization of Cts1p in vivo by a recombinant gene construct (pFRcts1-1) containing a C-terminal fusion to green fluorescent protein (Cts1p-GFP) in a myo1 strain partially confirms prior observations made by others with this construct [5]. Both the wild-type strain (MYO1 cts1Δ∷TRP1 pFRcts1-1) (Fig. 3A) and the myo1 strain (myo1Δ∷HIS5 cts1Δ∷TRP1 pFRcts1-1) (Fig. 3B) expressing the recombinant Cts1p-GFP fusion protein exhibit a localization of chitinase 1 between mother and daughter cells adjacent to the chitin primary septum. This result suggests that polarized secretion of chitinase 1 to the chitin primary septum was not prevented by myosin II deficiency. As expected for the wild-type strain, this polarized secretion is localized asymmetrically to the periplasmic space on the daughter side of the chitin septum (Fig. 3A) [5]. In contrast, chitinase 1 is delocalized in the myo1 strain encompassing both the daughter and the mother sides of the chitin septum (Fig. 3B). Quantitative densitometric analysis of these images shows a displacement of the Cts1p-GFP fluorescence signal to one side of the chitin septum in some cells (Fig. 1B, bottom left) and/or an increase in fluorescence intensity in others (Fig. 1B, bottom right), respectively. From this result we conclude that myosin II is important for maintaining the asymmetric localization of chitinase 1.

Fig. 3. Localization of secreted chitinase 1 in vivo.

Cellular localization of chitinase 1 (Cts1p) was achieved by expression of a GFP(S65T) fusion gene construct (pFRcts1-1) in (A) wild-type (MYO1 cts1Δ∷TRP1 pFRcts1-1) and (B) myo1 (myo1Δ∷HIS5 cts1Δ∷TRP1 pFRcts1-1) strain backgrounds both lacking the endogenous CTS1 gene [4]. Expression of the fusion protein was visualized by fluorescence microscopy performed on living cells using a 100× objective and a UV filter (Set I.D. 31001; excitation D480 nm/30×, emission D535 nm/40 m; from Chroma Technology). Digital images were captured and analyzed with a Spot RT monochrome cooled charged-coupled device digital camera (Diagnostic Instruments, Inc.). Densitometry was performed axially along the length of the fluorescence signal using the NIH Image program. Bar, 5 µm.

In conclusion, myosin II function is necessary to maintain normal regulation of chitinase 1 secretion levels during the cell cycle. A shift to a constitutive pattern of chitinase 1 secretion observed in the myo1 strain is not regulated at the transcriptional level. Furthermore, we present evidence that myosin II function is important to maintain the normal asymmetric localization of chitinase 1 in daughter cells undergoing cell wall separation.