Academic journal article Genetics

A Mutant Plasma Membrane Protein Is Stabilized upon Loss of Yvh1, a Novel Ribosome Assembly Factor

Academic journal article Genetics

A Mutant Plasma Membrane Protein Is Stabilized upon Loss of Yvh1, a Novel Ribosome Assembly Factor

Article excerpt

ABSTRACT

Pma1-10 is a mutant plasma membrane ATPase defective at the restrictive temperature in stability at the cell surface. At 37°, Pma1-10 is ubiquitinated and internalized from the plasma membrane for degradation in the vacuole. YVH1, encoding a tyrosine phosphatase, is a mutant suppressor of pma1-10; in the absence of Yvh1, Pma1-10 remains stable at the plasma membrane, thereby permitting cells to grow. The RING finger domain of Yvh1, but not its phosphatase domain, is required for removal of mutant Pma1-10 from the plasma membrane. Yvh1 is a novel ribosome assembly factor: in yvh1Δ cells, free 60S and 80S ribosomal subunits are decreased, free 40S subunits are increased, and half-mer polysomes are accumulated. Pma1-10 is also stabilized by deletion of 60S ribosomal proteins Rpl19a and Rpl35a. We propose that changes in ribosome biogenesis caused by loss of Yvh1 or specific ribosomal proteins have effects on the plasma membrane, perhaps by producing specific translational changes.

THE ribosome is a macromolecular assembly of four ribosomal RNAs and >80 different proteins. In eukaryotes, the ribosome is composed of a small 40S subunit and a large 60S subunit that perform the two critical activities of decoding messenger RNA and forming peptide bonds during translation. Ribosomes appear to play an even larger role than simply linking amino acids according to the genetic code. Ribosomes may influence post-translational events because ribosome- associated chaperones affect the folding of nascent polypeptides (Ito2005;Albanese et al. 2006).Moreover, recent evidence supports a model inwhich differences in ribosomal protein and RNA composition and modification of ribosomal protein and RNA produce heterogeneous ribosomes, differentially affecting translation of specific mRNAs (Mauro and Edelman 2002; Komili et al. 2007). In eukaryotic cells, the elaborate process of ribosome production begins in the nucleolus, continues in the nucleoplasm and cytoplasm, and is overseen by a growing list of up to 200 factors (Warner 1989; Henras et al. 2008).

We are interested in post-translational sorting mechanisms in the secretory pathway in Saccharomyces cerevisiae. These mechanisms are reflected in the disparate behavior of several different mutants of the polytopic membrane protein, Pma1. Many conformationally defective Pma1 mutants are recognized by an endoplasmic reticulum (ER) quality control mechanism shortly after synthesis and sent for ER-associated degradation (ERAD) (Nakamoto et al. 1998; Han et al. 2007). By contrast, other misfolded Pma1 mutants escape detection by ER quality control and are sent via vesicular transport into the secretory pathway (Pizzirusso and Chang 2004). One such mutant, Pma1-10, is delivered properly to the plasma membrane but fails to remain stable at the cell surface like wild-type Pma1. Instead, Pma1-10 is ubiquitinated and undergoes rapid endocytosis fromthe plasma membrane followed by vacuolar degradation (Gong and Chang 2001). Because Pma1 activity at the cell surface is essential for growth (Serrano et al. 1986), Pma1-10 is a temperature-sensitive mutant.

After detailed analysis of the behavior of Pma1-10 (Gong and Chang 2001), we reasoned that genetic selection for mutant suppressors of pma1-10 cells should reveal factors involved in recognition of misfolded protein at the cell surface. Indeed, loss of function in Yvh1, a tyrosine phosphatase, allows Pma1-10 to escape ubiquitination and remain stable at the plasma membrane (Liu and Chang 2006). Here, we report the unexpected finding that Yvh1 is a novel ribosome assembly factor. We suggest an effect of Yvh1 on ribosome composition and/or function may affect protein translation, leading to profound consequences at the plasma membrane.

MATERIALS AND METHODS

Yeast strains and media: KKX103-2B and -2D are pma1-10 yvh1 strains derived from the original suppressor of pma1-10 isolated following insertional mutagenesis, using a library generated by random insertions of LEU2 and lacZ; KKX15-1B (MATa yvh1TLEU2) was generated by backcross of KKX103-2D to L3852 (MATa his3Δ200 lys2D201 leu2-3,112 ura3-52 ade2). …

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