- Email: [email protected]
The yeast actin cytoskeleton
The y,east actin cytoskeleton Matthew
D Welch,
Douglas A Holtzman
University
of California,
and David G Drubin
Berkeley,
USA
Budding and fission yeast present significant advantages for studies of the actin cytoskeleton. The application of classical and molecular genetic techniques provides a facile route for the analysis of structure/function relationships, for the isolation of novel proteins involved in cytoskeletal function, and for deciphering the signals that regulate actin assembly in vivo. This review focuses on the budding yeast Saccharomyces cerevisiae and also identifies some recent advances from studies on the fission yeast Schizosaccharomyces pombe, for which studies on the actin cytoskeleton are still in their infancy.
Current
Introduction: cytoskeleton
the functions
Opinion
in Cell Biology
1994,
6:110-l
19
of the yeast actin
Although insights have been gained into the functions of the actin cytoskeleton of the budding yeast through the study of cytoskeletal mutants, its specific cellular functions remain elusive. The asymmetrical distribution of actin structures throughout the cell cycle (Fig. 1) suggests they have a role in directing new cell wall and membrane materials to growing regions of the cell. Cortical actin structures (referred to as patches) are located selectively at sites of surface growth: the bud, the septum [1,21 and the mating projection i3-61. Furthermore, cytoplasmic actin cables are oriented along the axes of formation of the bud [1,21 and the mating projection Ei-61.The phenotypes of cytoskeletal mutants (Table 1) indicate that the actin cytoskeleton functions in directing cell-surface growth. Mutations in the single essential actin gene (ACTI), and in other actin cytoskeletal genes, that disrupt the polarized organization of cortical actin structures result in an inability to undergo apical growth. The mechanisms through which actin participates in polarized cell-surface growth are not known. Defects in secretion, apparently in the late stages of the pathway (Golgi to plasma membrane) [71, are exhibited by act1 mutants, suggesting that actin might function in the movement of secretory vesicles. A direct role for actin in the movement of vesicles has yet ‘to be demonstrated. A&n-binding proteins that might mediate interactions between actin and secretory vesicles include Myo2p, a member of the dilute family of myosin I (unconventional or mini-myosin) proteins [81 that are thought to mediate vesicle movement along actin filaments, and tropomyosin, which regulates the
0
Actin,
fimbrin,
tropomyosin
Actin,
Abplp,
capping
I
protein,
cofilin,
fimbrin
0 1994 m Currm
Fig. 1. Organization
cerevisiae actin
and protein cytoskeleton.
components
Opinion in Cell Biolow
of the
Saccharomyces
interaction between myosin and actin in muscle cells and stabilizes actin cables in yeast cells [91. Both my02 [lOI and tpml (tropomyosin) ill’1 mutants accumulate membrane vesicles; however, defects in secretion are either not observed or are subtle. Identification of the origin of the membrane vesicles that accumulate in the mutants may help to further elucidate the roles of the MY02 and PM1 gene products in membrane trafficking. Recently, an important insight has been gained into one cellular function of actin. Kubler and Riezman [12*1 showed that actin and the actin-binding protein
Abbreviations ABP-actin binding protein; ACT-actin; ANC-actin RAH-reversion of act1 high osmolarity sensitivity; SLA-synthetic lethal with ABPl; SK-synthetic
110
0 Current
non-complementing; CAP-capping; SAC-suppressor of act1 temperature lethal with cap2; WY-suppressor
Biology
Ltd ISSN 0955-0674
COF-cofilin; MYO-myosin; sensitivity; SH-Src homology; of myosin; tpm-tropomysin.
The yeast actin cytoskeleton
‘abk
la. Genes involved
in function
of the hcchammyces
HOW
NUN
Cellular defects
Subcellular
lame
identitied
phenotype
in mutants*
localization
UPI
Biochemistry
Sequence
Holtzman
and Drubin
cerevisiae actin cyroskeleton.
;ene
CT1
Welch,
None detected
Lethal
homology
Cytoskeleton*” (Abpl p overproduction)
Actin patches
Morphology, cyioskeleton, secretion,
Cables and patches
Sequence similarities
References**
SH3 fsrc homology 3) domain, cofilinlike domain
I1 2’,36’,39*, 48.,771
Actin
osmoregulation, endocytosis, vesicle accumulation 4cr.7
sequence
lethal
n.d.
Actin-related
n.d.
proteins
1521
homology Genetics
tNC2
Genetics
n.d.
n.d.
n.d.
n.d.
136%39*1
tNC3
Genetics
n.d.
n.d.
n.d.
n.d.
136’,39’1
tNC4
Genetics
n.d.
n.d.
n.d.
n.d.
136’,39.1
3Pl
Biochemistry
ZAP2
Sequence homology
XT1
Biochemistry
WY01
Biochemistry
WY02
Genetics
Temperature sensitive
Morphology, Woskeleton, osmoregulation
Nucleus
4NCl
Morphology, cytoskeleton
Morphology. cytoskeleton
Actin patches
Morphology, cytoskeleton
Morphology, cytoskeleton
Actin patches
lethal
Cytokinesis, cell wall, osmoregulation*‘*’ Lethal
n.d.
Cytokinesis. cell wall, osmoregulation Morphology, cytoskeleton,
Human Enl and AF-9 proteins (implicated in acute leukemias)
Mammalian
capZ
[36*,39*]
129.,31,48*1
a-subunit
Aclin patches
n.d.““’
n.d.
Mamalian capZ bsubunit
129’,30,31, 42’,48’1
Low molecular weight severing proleins cofilin. actophorin, depactin, destrin
[27*,281
Myosin II (muscle myosin) heavy chain
I81
Myosin I (dilute family)
l8.10.11~ 58’1
vesicle accumulation
‘Not all phenotypes were evaluated for all mutants. **All references to each gene cited in the text are listed. For additional references, see literature cited within a given ,eference. References for ACT1 are too numerous IO list here. “‘The cellular defects were observed in cells with mutations in the indicated genes except in the case of the 48PJ gene, where the defects were seen when Abpl p was overproduced. *“The phenotype of a disruption mutation that removes the entire coding region has not been deponed. ““‘Although My01 p was reported to be located at the bud neck, this result has been called into question due to the observation that many non-affinity-purified ,abbit antisera will stain the bud neck. n.d. means not determined.
fimbrin (encoded by the SACG gene; [131> play an essential role in the internalization step of receptormediated endocytosis. The involvement seems to be direct, and not due to general defects in the secretory pathway, because a set (secretion) mutant with secretory defects similar to those of act1 mutants does not exhibit defects in endocytosis. Actin has also been shown to participate in endocytosis in epithelial cells [141. The powerful genetics available in yeast present a unique opportunity to study the role of the actin cytoskeleton in endocytosis in tivo. This review focuses on advances made in recent investigations into the cytoskeletal components of S. cerevisiae and S. pombe.
Actin structure/function S. cerevisiae provides two main advantages for the study of structure/function questions pertaining to actin. Firstly, a single, essential gene (AC73 encodes the sole source of ‘conventional’ actin in the cell (see also the section on actin-related proteins), thus eliminating concerns about isotype-specific functions and permitting the purification of biochemical quantities of a specific mutant actin. Yeast actin is 88% identical at the amino acid level to rabbit muscle actin [15,161, and is biochemically similar to it [17,18,19’1, suggesting that what is learned from studies in yeast will be generally
111
112
Cvtoskeleton
applicable. Secondly, homologous recombination can be employed to introduce the mutated version of the actin gene back into its chromosomal locus to assess its ability to function in viva. Several recent studies have used site-directed mutagenesis to address questions concerning actin structure and function in vivo and in vitro. One approach has been to target residues that have been previously implicated, either through biochemical crosslinking studies or studies on the crystal struchbk Gene name
PFYl
lb. Genes involved
in function of the Saccharomyces
HOW identified
Biochemistry, sequence homology
ture of monomeric actin, in specific aspects of actin function [19’,20,21*1. Rubenstein and colleagues have demonstrated the importance of the amino-terminal acidic residues of yeast actin for the interaction with myosin [19',21'1. In another study, Chen et al. 122'1 have examined the importance of the ‘hydrophobic plug’ in stabilizing actin filaments. According to the model proposed by Holmes et al. 1231 for filamentous actin, the hydrophobic plug accounts for the main interstrand contacts along the two-start helix (i.e two right-handed, intertwined helices). When a
cerevisiae actin cytoskeleton.
NUll
Cellular defects
Subcellular
phenotype
in mutants*
localization
very sick
Morphology, cytoskeleton
Diffuse cytoplasmic
Sequence similarities
Actin monomer binding protein
References*’
[49’.5cr,5
profilin
RAHl
Genetics
n.d.
nd.
n.d.
n.d.
132.1
RAHZ
Genetics
n.d.
r-cd.
n.d.
n.d.
1321
RAH3
Genetics
n.d.
n.d.
n.d.
132.1
5ACl
Genetics
Cold sensitive
Cytoskeleton Cytoskeleton, inositol auxotrophy
SAC2
Colgi and endoplasmic
No significant similarities
133.43.44’1
reticulum
n.d.
Cytoskeleton, secretion
n.d.
n.d.
1331
Cytoskeleton
n.d.
nd.
I331
SAC3
Genetics
n.d.
SAC4
Genetics
n.d.
n.d.
n.d.
n.d.
1331
SACS
Genetics
n.d.
n.d.
n.d.
n.d.
1331
SAC6
Genetics and biochemistry
Temperature sensitive
Morphology, cytoskeleton, endocytosis
Actin cables and patches
SAC7
Cold sensitive
Cytoskeleton
n.d.
SLA I
Temperature sensitive
Morphology, cytoskeleton
n.d.
Temperature sensitive
Morphology, cytoskeleton
n.d.
Actin filament bundling protein fimbrin No significant similarities St13 lsrc homology
II 2’.13,34. 36’,39’,40’ 42*.48-l I351
I4W
3) domains, sperm protein bindin
SLAZ
Genetics
SLCl
Genetics
n.d.
Morphology, cyloskeleton
n.d.
n.d.
142-l
SLCZ
Genetics
n.d.
Morphology, cytoskeleton
n.d.
n.d.
142.1
lPM1
Biochemistry
II
Temperature sensitive
Morphology, cytoskeleton, vesicle
Actin cables
Talin fcarboxyl terminal 200 amino acids)
Tropomyosin
140.I
[9,11’,36’, 390, 46’1
accumulation ‘Not all phenotypes were evaluated for all mutants. **All references to each gene cited in the text are listed. For additional references, see literature cited within a given reference. References for ACT1 are too numerous to list here. “‘The cellular defects were observed in cells with mutations in the indicated genes except in the case of the ABPt gene, where the defects were seen when Abplp was overproduced. “*CThe phenotype of a disruption mutation that removes the entire coding region has not been rewed. *****Although Myolp was reported to be located at the bud neck, this result has been called into question due to the observation that many non-affinity-purified rabbit antisera will stain the bud neck. n.d. means not determined.
The yeast actin cytoskeleton
charged residue (Let1266+A~p) was introduced into this region [22*1, yeast depending on this mutant actin showed a modest cold-sensitive growth defect. Significantly, in vitro studies showed that this mutation creates a change in the assembly properties of the actin monomer, including cold-sensitive defects in nucleation and filament elongation. An alternative approach to study structure/function relationships was carried out by Wertman et al. 124’1. Mutations were targeted to the surface of the protein by identifying clusters of two or more charged residues in the primary sequence; every charged residue in a cluster was replaced by alanine. The majority of the altered residues (81 o/o>are at or near the surface of the protein and are therefore unlikely to alter the tertiary structure of the protein. The resulting mutant actins were assayed in vivo by gene replacement. Sixteen conditional-lethal alleles were isolated, and many mutations corroborated predictions of the atomic model for actin filaments [24*,25,26’1. In addition, this collection has proven useful in dissecting the in vivo functions and biochemical properties of actin. For example, several mutations interfere with the organization of mitochondria in the cell, and another causes a defect in the interaction between actin filaments and the mushroom toxin phalloidin 126’1. Further study of actin mutants in budding yeast should result in additional insights into the relationship between actin structure and actin function.
Identification
of actin-binding
proteins
The functions of actin rely on the activity of a variety of actin-binding proteins. Although yeast cells do not exhibit behaviors such as motility that might necessitate a high degree of cytoskeletal complexity, it has become apparent that yeast is typical of eukaryotes in its complement of actin-binding proteins. Most classes of actin-binding proteins found in other eukaryotes have been identified in yeast (Table 1). Recently, Moon et al. 127’1 have identified a yeast homologue of the low molecular weight actin filament severing protein, cofilin, which is encoded by the COFl gene (this gene was also identified by Iida et al. 1281). Cofilin is an essential protein located at cortical actin structures. In addition, Moon et al. have identified three more activities in yeast extracts that may represent other actinbinding proteins 127’1.
et al. 1291 have identified a homologue of the a-subunit of vertebrate capping protein (encoded by the CAP1 gene). Capping protein functions as a heterodimer of one a-subunit and one p-subunit (encoded by CAp2, previously identified in 13011,and is located at cortical actin structures 1311.Deletion of one or both of the genes encoding the a- and p-subunits causes aberrant cell morphology, disorganization of cortical actin structures and partial disappearance of cytoplasmic actin cables 129’,3Ol. Therefore, capping protein might function to nucleate or stabilize actin cables and other
Amatruda
Welch,
Holtzman
and Drubin
actin filament structures in vivo, a role that would be consistent with its biochemical activity in vitro.
Genetic identification cytoskeletal function
of proteins
important
for
Genetic techniques have been used to identify a variety of additional genes that are important for actin cytoskeletal function in yeast. The significance of using novel genetic approaches is underscored by the observation that many of these genes have not been identified through biochemical methods in other organisms. Using mutant alleles of the ACTI gene, two novel avenues were taken to identify genes important for actin function. Chowdhury et al. 132’1 selected for mutations that suppress a specific phenotype of act1 mutants, sensitivity to high osmolarity (osmosensitivity). Previously, mutations in the SACl-SAC7 genes were identified as suppressors of the temperature sensitivity of act1 mutants 133351. Chowdhury et al. identified three genes (IUHl, RAH2 and RAH.. reversion of act1 high osmolarity sensitivity), one of which (Z?&f..) is known not to be allelic with any previously identified cytoskeletal gene. Mutations in &I./Y3 cause defects in the organization of cortical actin structures, indicating that Rah3p, its protein product, is important for actin cytoskeletal function. In another study, Welch et al. 136’1 screened for extragenic mutations that fail to complement the temperature-sensitive phenotype exhibited by act1 alleles, a technique that has been used to identify genes that encode physically interacting proteins 137,381. At least four new genes, ANCl-ANC4 (actin non-complementing), were identified. Mutations in these genes interact genetically with mutations in genes for a&-i-binding proteins 139’1, providing evidence that the proteins participate in cytoskeletal function. Furthermore, mutations in AhCl cause defects in actin organization, suggesting that Anclp is important for cytoskeletal function 136’1. Another approach to identify cytoskeletal genes has been screening for mutations that exhibit specific genetic interactions with alleles of genes encoding actinbinding proteins. Holtzman et al. 140’1 identified SLAl and SLA2 (synthetic lethal with a&l) (and rediscovered SACG, which encodes the actin-binding protein fimbrin 1131) by screening for mutations that are lethal in combination with a null mutation in ABPl (actinbinding protein; Table la>. Slalp, like Abplp, has SH3 domains (SIC homology domain 31, which are present in other membrane cytoskeletal proteins and may link the cytoskeleton to intracellular signaling pathways 1411.Sla2p has a 200-amino-acid carboxy-terminal domain that is similar to the carboxy terminus of talin, a component of Iibroblast focal adhesions. Mutations in both X41 and SLA2 cause unique defects in the organization of cortical actin structures, indicating that each protein plays a different role in the
113
114
Cvtoskeleton
functions of the cortical cytoskeleton. Using a similar approach, Karpova et al. [42*1 isolated mutations in SACG and in two additional genes (SLCl and SLCZ synthetic lethality with cap2) by screening for mutations that are lethal in combination with a null mutation in the CAP2 gene. Defects in cytoskeletal organization are exhibited by slcl and slc2 mutants, indicating that Slclp and Slc2p are involved in actin function. It is now important to determine the intracellular location of the RAHl-3 ANCl-4, $31-2, SLCl-2 gene Jducts, and the molecular nature of the interaction between these proteins and actin. Genetic approaches have sometimes led to the identification of genes whose functional relationship to actin remains difficult to elucidate, although this does not diminish the importance of the relationship. Mutations in SAC1 were identified as suppressors of act1 mutations 1331, and were subsequently found to suppress defects caused by loss of the phosphatidylinositol/phosphatidylcholine transfer protein, Secl4p [431, suggesting that Saclp has a function in secretion and/or membrane metabolism. Whitters et al. [44'1 have recently demonstrated that Saclp is an integral membrane protein located in the endoplasmic reticulum and Golgi, and that sac1 mutants have an unusual inositol auxotrophy. These results suggest that Saclp is likely to function in inositol glycerophospholipid metabolism, and raise the question of the mechanisms through which actin and Saclp interact. One potential mechanism is through a&n-binding proteins whose activities may be regulated by phospholipids. A connection between phospholipid metabolism and actin cytoskeletal function has been suggested by biochemical studies 145-471. Genetic analysis provides one avenue towards testing the in z&o relevance of these interactions.
Genetic function
analysis of a&n-binding
protein
Actin-binding proteins with a variety of biochemical activities must act together to produce a functional cytoskeleton. Issues such as whether certain subgroups of actin-binding proteins work together in tivo can be addressed by identifying genetic interactions between mutations in different genes coding for actin-binding proteins. To this end, several groups l11’,39’,40’,42*,48*1 have examined the consequences of making combinations of null mutations in genes that encode the yeast actin-binding proteins Abplp, fimbrin, tropomyosin, capping protein and Myo2p, as well as in other genes implicated in cytoskeletal function. Specific pair-wise combinations of null mutations resulted in lethality. Surprisingly, none of these pairs of genes encode proteins with similarity in structure or biochemical activity. This suggests that although these proteins may have functional homologies, their effects on the cytoskeleton are achieved through different mechanisms.
Genetic studies have also addressed the functions of the actin monomer binding protein profilin. It has been suggested that this protein participates in multiple (and perhaps related) processes, including actin cytoskeletal function and PIP2 (phosphatidylinositol 4, 5bisphosphate)-mediated signaling [46,471, yet its role in vivo remains uncertain. A report by Magdolen et al. [49*1 showing that overproduction of profilin can suppress the lethality caused by overproduction of actin suggests that, at least under certain conditions, profilin may act in vivo to sequester monomeric actin. A promising avenue for the elucidation of profilin function is the creation of site-directed mutations that affect specific activities of profilin. Using this approach, Haarer et al. 150’1 have created mutations that affect actin and/or PIP2 binding. Analysis of these mutants suggests that the actin-binding property of profilin is important for its in vivo function. Questions regarding the relevance of PIP2 binding in vivo remain unresolved (see also 1511).
Actin-related interactions
proteins
and actin-microtubule
In general, actins from different species are greater than 70% identical in amino acid sequence, with the primary amino .acid sequence of S. cerevisiae actin being 88% identical to mammalian actins. Interestingly, a family of actin-related proteins that are 4+50% identical to actin in their amino acid sequences have been discovered in S. cerevisiae (AcQp 15211,Schizosaccbaromyces pombe (act2p 1531) and in mammalian cells (actin-RPV [541, centractin [55,561, actin [571). The S. cerevisiae ACT2 and S. pombe act2 genes are essential, but their cellular functions have yet to be determined. Vertebrate actin-related proteins are involved in microtubule-based vesicle motility 154-571, and by analogy, the yeast proteins may perform similar functions. Determining the cellular location of Act2p and identifying proteins that interact biochemically and genetically with Act2p in yeast will increase our understanding of the functions of actin-related proteins in eukaryotic cells. Another instance of possible convergence between the functions of actin and microtubules is the finding by Lillie and Brown 158’1 that overproduction of Smylp @MYI = suppressor of myosin), which shares sequence similarity with the motor domain of the microtubule-based motor protein kinesin, can suppress mutations in the MY02 gene, which encodes a myosin I actin filament-based motor protein. Although deletion of SMYl causes no detectable phenotype, the deletion is lethal in combination with a MY02 mutation, providing more evidence that Smylp and Myo2p proteins perform similar functions in vivo. The sequence similarities to motor proteins suggest that Smylp and Myo2p might be important for vesicle transport, and present the interesting possibility that the actin and mi-
The yeast actin cytoskeleton Welch, crotubule cytoskeletons in this respect.
perform
redundant
functions
Another cellular process in which both actin and microtubules participate is the alignment of the mitotic spindle. Before anaphase, the mitotic spindle is positioned with one spindle pole at or through the mother-bud neck, allowing proper chromosome segregation during anaphase. Palmer et al. WI showed that spindles are misoriented in act1 mutants and in mutants with disrupted astral microtubules, suggesting that astral microtubules interact with the actin cytoskeleton to orient the spindle. This is consistent with the finding that pharmacological disruption of the actin cytoskeleton in Caenorhabditis elegans blastomeres changes the position of the spindle 160,611, and that actin function is necessary for migration of the nucleus to the tip of the mating projection in S. cerevisiue [61.
Signaling/cell
cycle control
of actin organization
The asymmetric disposition of actin in S. cerevisiae is one cellular readout of complex genetic pathways that influence morphogenesis, bud site selection and the formation of the pheromone-induced mating projections. (For a comprehensive review of the genetic control of cell polarity in Succbaromyces, see 162X341). Several recent papers have begun to explore the signaling pathways that control the rearrangements of the yeast actin cytoskeleton. Given the rapid cytoskeletal reorganizations that are possible in a variety of cell types (1651 and references therein), it is certain that post-translational modifications and direct modulations by second messenger molecules are central to the regulation of actin assembly in vivo. Using a variety of mutants and misexpression strategies, Lew and Reed 16@1have addressed the role of the Cdc28 kinase in the regulation of morphogenesis and the localization of cortical actin structures throughout the cell cycle. Importantly, they find that alterations in the expression of Gl and G2 cyclins have dramatic effects on polarization of the cytoskeleton. In addition, reorganization of actin following the downshift of a temperature-sensitive cdc28 mutation was found to be independent of de novo protein synthesis, suggesting that phosphorylation plays a direct role in the regulation of the yeast actin cytoskeleton. It is now important to determine whether any cytoskeletal components in yeast are direct targets for the Cdc28 kinase. Calcium has well documented effects on the activity of several actin-binding proteins isolated from motile cells (see 1651 for examples), and it is a common second messenger molecule generated in response to diverse cell-surface signals. Yeast contain a single essential gene (CMDI) that encodes calmodulin, a high affinity calcium-binding protein implicated in diverse cellular functions 167,681. Calmodulin has now been immunolocalized in S. cerevisiae and found associ-
Holtzman
and Drubin
ated with sites of surface growth throughout the cell cycle 169’,701, with the pattern of calmodulin localization that is overlapping but distinct from that of actin. Interestingly, mutations in calmodulin can affect the localization of actin 163’1 (Y Ohya, personal communication) suggesting that calcium signaling mechanisms may function in the polarization of the yeast actin cytoskeleton.
CDC42, a gene involved in development
of cell polarity and asymmetric assembly of the actin cytoskeleton in S. cerevisiue, is a member of the rho family of ras-related small GTP-binding proteins 171,721. Matsui and Toh-e have now identified two new members of this family, RHO3 and RHO4 1731, and have demonstrated their involvement in development of cell polarity 174.1. Strains lacking RHO3 and RHO4 have defects in morphogenesis and delocalized actin cytoskeletons. Additionally, Chenevert et al. 1751find that the growth defects of the rho.3 strain can be suppressed by overexpression of either CDC42 or BEMl, an SHIcontaining gene involved in polarity development . The identification in mammalian cells of an SH3 ligand with homology to rho-GTPase activator proteins 1761has generated interest in the link between yeast SH3 proteins (Abplp, Bemlp, Slalp and Rvs167p; 140’,75,77,78*1) implicated in morphogenesis and actin cytoskeleton organization, and the other proteins that control cell polarity. In addition, the recent discovery that rho proteins control actin stress fiber assembly 1791 in Iibroblasts highlights the emerging parallels between the regulation of the actin cytoskeleton in yeast and in more complex cells.
Schizosaccharomyces
pombe
While studies on the actin cytoskeleton in fission yeast are in their infancy compared with those in budding yeast, similar advantages are afforded by S. pombe for addressing the function of the actin cytoskeleton. It will be useful to compare and contrast cytoskeletal regulation in these two yeasts as a way to identify conserved mechanisms important for cytoskeleton function. S. pombe is a particularly promising organism for elucidation of the mechanism and regulation of cytokinesis, because cytokinesis appears more similar to the process in vertebrate cells 180,811, because a large number of mutants defective in cytokinesis are available 1821, and because cell-cycle regulation in S. pombe is well understood 1831. Balasubramaman et al. t84.1 have now shown that the cdc8 gene of S. pombe encodes a novel tropomyosin essential for cytokinesis, and have localized this tropomyosin to small actin patches as well as to the medial band of filamentous actin that forms at the septum. In addition, antibody cross-reactivity was found in S. pombe against transgelin, a widely distributed actin filament gelation factor which is downmodulated in mammalian cells by transformation and non-adherent culture conditions 185’1.
115
116
Cvtoskeleton
.
Conclusions
The past year and a half has seen a sharp rise in activity in research on the yeast actin cytoskeleton. Many new genes have been identified and their in vivo roles tested, and the potential for identifying regulatory interactions and structure-function relationships is being realized. Because a concerted effort by a large research community is being focused on understanding virtually every aspect of the biology of the simple yeast cell, we can look forward to the development of an integrated view of cytoskeletal function and regulation.
References
and recommended
. ..
of particular interest, published have been highlighted as: of special interest of outstanding interest
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2.
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HASEK J, RUPES I, SVOUODOVA J, STREIDLOVA E: Tubulin and Actin Topology during Zygote Formation of Saccharvmyces cemvfsfhe. J Gen Mfcrobfol 1987, 133: 3355-3363.
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READ EB, OKAMURA HH, DRUUIN DG: Actin- and ‘I’ubulin-Dependent Functions during Saccbaromyces cerwistae Mating Projection Formation. Mol Bfol Ceil 1992, 3:429-444.
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H: Actin Step of
and Fibrin Endoeytosis
Arc Required for in Yeast. F&Z0 J
is improteins
13.
reading
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4.
This is the first demonstration that the yeast actin cytoskeleton ponant for endocytosis. The role of individual actin-binding in endocytosis is also explored.
of Yeast In Vfw.
of a Split Yeast Gene: of the Actin Gene in Nut1 Amd Sci USA 1980,
A: Yeast Actin is Relatively 1992, 206:949-955.
Well
Be-
COOK RK, BLAKE WT, R~JHENSIF.IN PA: Removal of the Amino Terminal Acidic Residues of Yeast Actin. J Biol Cbem 1992, 267:943&9436. This study is a good example of the utility of combining sitedirected mutagenesis with In vfw and In vitro studies. The removal of the amino-terminal charges from yeast actin was shown to interfere with the intewction between actin fiiarnents and myosin, although these mutant actins were still capable of supporting growth when expressed from a centromere-based plasmid. 19. .
20.
JOHANNt? FJ, GALLWIIZ D: Site-Directed Mutagenesis Yeast Actin Gene: a Test for Actin Function In Vfw. J 1991, 10:3951-3958.
of the EMBO
21. .
CWK RK, ROOI D, MILLER C, HEISLER E, RUI~ENSI%IN PA: Enhanced Stimulation of Myosin Subfragment 1 ATPase Activity by Addition of Negatively Charged Residues to the Yeast Actin NH2 Terminus. J Bfol Cbem 1993, 268:241&2415. In a complementary study to I191, addition of negative charges to the amino-terminus of actin was shown to increase the ability of actin filaments to stimulate the ATPase activity of rdbbit muscle myosin Sl fragments. 22. .
CHEN X, COOK RK, RUBENSTEIN PA: Yeast Actin with a Mutation in the Hydrophobic Plug Between Subdomains 3 and 4 (La&) Displays a Cold-Sensitive Polymerization Defect. J Cell Bfol 1993, 123:118%1195. As a test of the structural model for F-actin [see 231, an aspanic acid was substituted for a leucine in a stretch of hydrophobic residues thought to make important contacts between the two strands of the actin filament. This mutant actin shows a cold-sensitive polymerization defect In vftro, providing further support for the Holmes model for the actin filament. 23.
24. .
HOLMES KC, Parr D, GEISHARD W, KAU~CH W: Atomic of the Actin Filament. Natme 1990, 347:44-i9.
IModel
WERI’MAN KF, DR~JI~IN DG, DO~WEIN D: Systematic Mutationai Analysis of the Yeast ACT1 Gene. Gznetfcs 1992, 132:337-350. Mutations in ACT1 were successfully targeted to the surface of the actin monomer using a charged-to-aianine replacement strategy, resulting in a large collection of conditional alleles. These mutants have proven useful for the examination of structure/function questions relating to the actin filament, as well as for an analysis of the role of actin In vfw [see 26’1.
The yeast actin cytoskeleton 25.
WERTMAN the Right
KF. DRUBIN DG: Actin to Assemble. Sc&zce
Constitution: Guaranteeing 1992, 258:759-760.
Welch,
Holtzman
and Drubin
117
DRUUIN DG, JONES HD, WERTMAN KF: Actin Structure and Function: Roles in Mitochondrial Organization and Morphgenesis in Budding Yeast and Identification of the PhalloidinBinding Site. Mot Bfol Cell 1993. 4:1277-1294. Mutations in ACT1 identify a role for actin in the organization of mitochondria within the cell, and implicate specific residues in the interaction with phdlloidin.
VINH DBN, WELCH MD, Cow AK, WERTMAN KF, DRUBIN DG: Genetic Evidence for Functional Interactions Between Actin Noncomplementing (AX) Gene Products and Actin Cytoskclctal Proteins in Succbammyces cemvlslae. Genettcs 1993, 135:275-a. Mutations In ANCgenes (see l36.1) are shown to inter-act genetically with mutations In actin biding protein genes, providing strong evidence that the Ant gene products participate in cytoskeletal function. Also, genetic models for extragenic noncomplementation are explored.
27. .
40. .
39. .
26. .
MOON AL, JANMEY PA, LOUIE KA, DRUBIN DG: Cofilin is an Essential Component of the Yeast Cortical Cytoskclcton. / Cell Bfol 1993, 120:421-435. Cofilln is the first actin filament severing protein identified in yeast. CotilIn is shown to be an essential protein that is located at cortical actin structures. 28.
IIDA K, MORIYAMA K, MATSUMOI’O S, KAWASAKI H, NISHIDA E, YAHARA I: Isolation of a Yeast Essential Gene, COFI. that Encodes a Homologue of Mammalian Cofilin, a LowM(r) Actin-Binding and Depolymcrizing Protein. Gene 1993, 124:115-120.
AMAIR~JDA JF, GAITERMEIR DJ, KA~~~OVA TS. COOPER JA: Effects of Null IMutations and Overexpression of Capping Protein on Morphogcnesis, Actin Distribution and Polarized Sccrction in Yeast. / Cell BIol 1992, 119:1151-1162. The cloning of the a-subunit of yeast capping protein is reported, and the cellular roles of capping protein are explored through the phenotypes of mutants.
29. .
30.
AMXIHWA
JF, CANNON JF, TATCHELL K. HUC C, Ccor~~ Disruption of the Actin Cytoskcleton in Yeast Capping tein IMutants. Nartrre 1990, 344:352354.
JA: Prcl
31.
AMAIRUDA JF, COOPER JA: Purification, Characterization, lmmunofluoresccnce Localization of Succbarumyces vLslae Capping Protein. J Cell Btol 1992, 117:1067-1076.
and ten+
CHOWDHURY S, SMITH KW, GUS~N MC: Osmotic Stress and the Yeast Cytoskeleton: Phenotype-Specific Suppression of an Actin IMutation. J Cell Btol 1992, 118:561-571. This report demonstrdtes that the actin cytoskeleton is sensitive to osmotic stres% Suppressors of the osmosensitive phenotype of act1 mutants were isolated. These reside in three genes, one of which \vaS shown to be novel and important for actin cytoskeletal function. 32. .
33.
NOVICK P, OSM~NV BC, B~IJTEIN D: Suppressors Actin Mutations. Generics 1989, 121:659-674.
of Yeast
34
ADAMS AE, DO-IXXIN Actin Mutations that iu- 1989, 121:675-683.
of
35.
DUNN
TM,
SHORTLE
Temperature-Sensitive
cenwlsiae. Mol Cell
D: Arc
Dominant Reciprocally
Suppressors Suppressed.
SXARNS T, &XSIFIN D: Unlinked lation of New Conditional-Lethal Tubulin Genes of Saccbaromyces 119:24%260.
Noncomplcmentation: Iso Mutations in Each of the cerwlslae. Generics 1988,
HAYS Ts, DEUHING R, ROBERISON B. PROUI’ M, FULLER MT: Interacting Proteins Identified by Genetic Interactions: a IMissense *Mutation in Alpha-Tubulii Fails to Complement Alleles of the Testis-Specific Beta-Tubulin Gene of Lhwopblla
melanogaster
KOCH CA, ANDERSON D, MORAN MF, ELLIS C, PAWSON T: SH2 and SH3 Domains: Elements that Control Interactions of Cytoplasmic Signaling Proteins. Science 1991. 252668-674.
42. .
Mutations that EnIn Saccbarumyces Morphogcncsis, I35:693709. screen for mutations the capping protein were shown to be
43.
CI.EVFA AE, NOVICK PJ, BANKAI’IX VA: Mutations in the SAC1 Gene Suppress Defects in Yeast Golgi and Yeast Actin Function. J Cell Biol 1989, 109:2939-2950.
KARPOVA TS, LEP~T MM, COOPER JA: hance the cap2 Null Mutant Phenotype cemvfsfae Affect the Actin Cytoskclcton, and Pattern of Growth. Generics 1993, Two genes (.%X1 and SLCZ) were identilied in a that are lethal in combination with a null allele of gene CAP2. The SLCl and SLC2 gene products Important for cytoskeletal function.
44. .
WHI~RS EA. CLEVES AE. MCGEE Tp, SKINNER HB, BANKAIIX VA: SAClp is an Integral Membrane Protein that Influences the Cellular Requirement for Phospholipid Transfer Protein Function and lnositol in Yeast. J Cell Bfol 1993, 122:79-94. Saclp, originally identified in a screen for suppressors of an actin mutation, is shown to be an integral membrane protein, resident in the endoplasmic reticulum and Golgi. Consistent with previous findings that Saclp functionally interacts with a phospholipid transfer protein (Secl4p), evidence is presented that Saclp is likely to function in inositol glycerophospholipid metabolism. 45.
LAS~INC I, LINDBERC U: Specific phatidylinositol 4,5Bisphosphate 1985, 314:472-l74.
46.
GOLDSCHMIDT CP, MACHESKY LM, BALDASSARE JJ, POLLARD TD: The Actin-Biding Protein Prohlin Binds to PIP2 and Inhibits Its Hydrolysis by Phospholipase C. Science 1990, 247:1575-1578.
47.
GOLDSCHMIDT CP, KIM JW, MACH-KY LM, RHEE SG, POLLARD TD: Regulation of Phospholipase C-y 1 by Prolilin and Tyrosine Phosphorylation. Science 1991, 251:1231-1233.
D: Null AIlclcs of SAC7 Suppress Actin Mutations in Succhammyces Biol 1990, 10:2308-2314.
WELCH MD, VINH DBN, OKAMUKA HH, DRUBIN DG: Screens for FXtragenic Mutations that Fail to Complement acrl AIlclcs Identify Genes that arc Important for Actin Function in &zccbarvmyces cemuisiae. &wettc.s 1993, 135:265-274. This report demonstrdtes that mutations in genes for actin-binding proteins fail to complement a mutant allele of ACTl. Screens for extragenic noncomplementing mutations led to the isolation of genes that are important for actin function (see also I39.1).
38.
41.
Yeast Cener-
36. .
37.
HOLTZMAN DA, YANG S, DRUBIN DG: Synthetic-Lethal lnteractions Identify Two Novel Genes, SLAI and X42, that Control Membrane Cytoskclcton Assembly in Sac&aromyces cerevisiae. J Cell Blol 1993, 122:635-644. The novel genes (SLAI and S~z) with sequence similarity to proteins involved in cortical cytoskeletal function were identified in a Screen for mutations that are lethal in combination with a null allele ln the Abel gene. SL42 and SLA2are shown to participate in unique aspects of membrane cytoskeletal function in L&O.
Mel Cell Bid 1989, 9:875-884.
Interaction Between Phosand Profilactin. Nartrre
ADAMS AEM. CARPER JA, DRUBIN DG: Unapcctcd Combinations of Null Mutations in Genes Encoding the Actin Cytoskeleton Are Lcthal in Yeast. Mol Btol Cell 1993, 4:45%468. Potential interactions between subgroups of actin-binding proteins in vlw were explored by identifying genetic interactions between mutations in genes for actin-binding proteins. 48. .
MACDOIEN V, DRUIXN DG, MAGI G, BANDLOW W: High Levels of Profilin Suppress the Lethality Caused by Ovcrproduction of Actin in Yeast Cells. FEBS kft 1993, 316:4147. This report suggests that, under certain conditions, p&din might act to sequester monomeric actin In ufw. 49. .
50. .
HAARER BK, PIXOLD Yeast Prolilin. Mol
AS, BROWN
SS: IMutational
Cell Blol 1993 13:7864-7873.
Analysis
of
118
Cytoskeletop Sitedirected mutagenesis of profilin presents a promiiing avenue for correlating the biochemical properties of purihed profilin fn titro with the function of proiilins tn uiuo. 51.
VOJTEK A, HMRER B, FIELDJ, GER~TJ, POLLARDTD, BROW~N S, WICLER M: Evidence for a Functional Link Bctwcen Rotilin and CAP in the Yeast S. cerwtikae. Cell 1991, 66497-50s.
in cyclin expression affect morphogenesis and the localization of cortical actin structures. 67.
DAVIS TN: Mutational Analysis of Calmodulin in Sac&arvmyms cmklae. Cell Galctzrm 1992, 13:435-444.
68.
OHYA Y, ANRAKU Y: Yeast Calmodulin: Structural and Functional Elements Essential for the Cell Cycle. Cell Calcium 1992, 13:445-i55.
52.
.%HWOB E, MARTIN RP: New Yeast A&n-Like Gene Rcquircd Late in the Cell Cycle Nalnre 1992, 355:179-182.
53.
LEES MJ, HENRY G, HELFMANDM: Identification of a&7, an EsscntiaI Gcnc in the Fission Yeast Scbfxasaccbammyces pombe that Encodes a Protein Related to Actin. Proc Nat1 Acad Scf USA 1992, 8980-83.
54.
LEES MJ, HEIEMAN DM, SCHROERTA: A Vcrtcbratc ActinRelated Protein Is a Component of a Multisubunit Complex Involved in Microtubule-Based Vesicle Motility. Nature 1992, 359244-246.
55.
CLARK SW, MEYER DI: Ccntractin is an Actin Homologuc Associated with the Ccntrosomc. Natrrre 1992. 359:246250.
56.
PASCHAL BM, Houluutt EL, PFI.!X+R KK, CLARK S, MEYER DI, VALLEE RB: Characterization of a S@kDa Polypcptidc in Cytoplasmic Dyncin Preparations Rcvcals a Complex with pl5OGLUED and a Novel Actin. / Blol Cbem 1993, 268:1531%15323. TANAKA T, SHIBA~AKI F, ISHIKAWA M, HIRANO N, SAKAI R, NISHIDA J, TAKENAWA T, HIMI H: Molecular Cloning of Bovine Actin-Like Protein. Actin 2. Blocbem Biophys Res Commtcn 1992, 187:1022-1028.
74. .
57.
DWE SH, BROWN SS: Suppression of a Myosin Defect by a 58. . Kincsin-Related Gene. Nature 1992, 356:3%-361. Proteins with sequence similarity to microtubule and actin based motor proteins perform overlapping functions In f&o. PALMER RE, SULLIVAN DS, HUFFAKERT, KOSHUND D: Role of Astral Microtubules and Actin in Spindle Orientation and Migration in the Budding Yeast. Saccbarvmyces cenwfsfae. J Cell Btol 1992, 119:583593. Mitotic spindles are m&oriented in act1 mutants, suggesting that the actin cytoskeleton Interacts with microtubules to orient the spindle. 59. .
60.
61.
HILL DP, SHAKES DC, WARD S, S’IROMES: A Sperm-Supplied Product Essential for Initiation of Normal Embryogcncsis in Ciaenorbabdftls eiegans Is Encoded by the Paternal-Effect Embryonic-Lethal Gcnc, spell. L%v Bioll989, 136:154-166. HIU DP, STROME S: Brief Cytochalasin-Induced Disruption of Microtilamcnts during a Critical Interval in 1Ccll C. e@ans Embryos Alters the Partitioning of Developmcntal Instructions to the 2Ccll Embryo. Deuelopment 1990, lOft:159-172.
62.
DRUBIN DG: Dcvelopmcnt of Cell Polarity in Budding Yeast. Cell 1991, 65:109%1096.
63.
CHANTJ, P~UNCLEJR: Budding and Ccl1 Polarity in Saccbammyces cen?vLslae. Cm-r QDin Genet Dev 1991, 1342-350.
64.
MADDEN K, COS~CAN C, SNYDERM: Cell Polarity and Morphogencsis in Saccbaromyces cerwisiue. Trena3 Cell Biol 1992, 2:22-29.
65.
STOSSELTP: On the Crawling of Animal Cells. Science 1993. 260:1086-1094.
LEW DJ, REED SI: Morphogcncsis in the Yeast Cell Cycle: Regulation by Cdc28 and Cyclins. J Cell Btol 1993, 120:130>1320. This study begins to dissect the cell cycle dependent organization of the actin cytoskeleton through the use of temperature-sensitive alleles of WCZ8and ectopic expression of Gl and G2 cyclins. Changes
BROCKEI~HOFF SE, DAVIS TN: Calmodulin Concentrates at Rcgions of Cell Growth in Succbarvmyces cemvlslae. J Cell Btol 1992, 118:619629. Immunolocalization of calmodulin in yeast shows that it is associated with sites of surface growth Isee also 701, and analysis of mutants suggests that localization of actin and calmodulin may be interdependent.
69. .
70.
SUN G-H, OHYA Y, ANRAKU Y: Yeast Calmodulin Localizes to Sites of Cell Growth. Protoplasma 1992, 166:110-113.
71.
ADAMS AE, JOHNSONDI, LONCNECKERRM, SLOA’I’BF, PRINCLE JR: CDC42 and CLX43, Iwo Additional Genes Involved in Budding and the Establishment of Cell Polarity in the Yeast Saccbaromyces cerwfslae. J Cell Bfol 1990, 111:131-142.
72.
JOHNWN DI, PRINGLE JR: Molecular Characterization of CDc42, a Saccbaromyces cetwfsfae Gene Involved in the Development of Cell Polarity. J Cell Btol 1990, 111:14%152.
73.
MA’IXUI Y, TOH-E A: Isolation and Characterization of Two Novel rus Superfamily Genes in Saccbaromyces cetwidae. Gene 1992, 114:43-49.
MA-IXJI Y, TOH-E A: Yeast RHO3 and RHO4 rus Superfamily Gcncs arc Ncccssary for Bud Growth, and Their Defect is Suppressed by a High Dose of Bud Formation Genes CLX42 and BEMI. Mol Cell Biol 1992, 12:5690-5699. The CLX42and BEMl genes implicated in cell polarity development were shown, when overexpressed, to suppress rho3 mutations, suggesting that the RHG3 gene product may function in the same pathway as the CDC42 and BEMl gene products. 75.
CHENEVERTJ, CORRADOK, BENDERA, PHINGLEJ, HERSKOWI’I’Z I: A Yeast Gene (BEMI) Necessary for Cell Polarization Whose Product Contains Iwo SH3 Domains. Nuttrre 1992, 356:77-79.
76.
CICCH~XI P, MAYER BJ, THIEL G, BAL~MORE D: Identification of a Protein That Binds to the SH3 Region of Abl and Is Similar to Bcr and GAPrho. Scfence 1992, 257:803-806.
77.
DRUBIN DG, MULHOLLANDJ, ZHIMIN 2, BOTS~FIN D: Homology of a Yeast Actin-Biding Protein to Signal Transduction Proteins and Myosin-I. Nature 1990, 343:288-290.
78. .
BAU~R F, URDACI M, ANGLEM, CROUX~ M: Alteration of a Yeast SH3 Protein Leads to Conditional Viability with Dcfccts in Cy-toskclctal and Budding Patterns. Mol Cell Biol 1993, 13:507c&5084. Isolation of mutants that fail to survive stationary phase identified RVS167, a gene that contains an SH3 domain. Cells containing mutations in RVSl67also showed defects in morphogenesis and altered bud-site selection. 79.
RIDLEY AJ, HALL A: The Small GTP-Binding Protein rho Regulates the Assembly of Focal Adhesions and Actin Stress Fibers in Response to Growth Factors. Cell 1992, 70389-399.
80.
ALFA CE, HYAMS JS: Distribution of Tubulin and Actin through the Cell Division Cycle of the Fission Yeast Scbfzosaccbaromyces japonfcus var. wrsatilis: a Comparison with Scblzosaccbarvmyces pombe. J Cell Sci 1990, 96:71-77.
81.
J~HZHOVAJ, RUPF~I, S’rat%~~ovri E: F-Actin Contractile Rings in Protoplasts of the Yeast ScbizosaccbaFomyces. Cell Biol Int Rep 1991, 15607-610.
66. .
The yeast actin cytoskeleton 82.
MARKS J, FANKHAUSER C, SIMANIS V: Genetic Interactions in the Control of Septation in Sc&izosucc&uromycespombe. J CelI Sci 1792, 101801-808.
83.
NURSE P: Universal Control Mechanism Regulating Onset of M-Phase. Nature 1990, 344:503-508.
84.
BAIAWIUIAMANIAN
MK,
HELFMW
DM,
HEMMINGSEN
SM:
Welch,
Holtzman
and Drubin
Shape Change Sensitive ActinGelling Protein. J Cell Bfol 1993, 121:1065-1073. Immunoblots using antibodies against transgellin identified cross-reactivity in S. pombe.
A
New Tropomyosin Essential for Cytokincsis in the Fission Yeast S pombe. Nature 1332, 36084-87. Cloning and sequencing of cdc8 identified a novel tropomyosin in S. pornbe. Immunolocalization of cdc8p showed that it is associated with both the actin patches and the medial hand of actin that forms during cytokinesis. .
85.
.
SHAPLAND
C,
HSUAN
tion and Properties
JJ, TOI-rv NF, LAWSON D: Purificaof Transgclin: A Transformation and
MD Welch, DA Holtzman and DG Drubin, Department of Molecular and Cell Biology, 455 Life Sciences Addition, University of California, Berkeley, CA 94720, USA.
Author for correspondence:
DG Drubin.
119
D Welch,
Douglas A Holtzman
University
of California,
and David G Drubin
Berkeley,
USA
Budding and fission yeast present significant advantages for studies of the actin cytoskeleton. The application of classical and molecular genetic techniques provides a facile route for the analysis of structure/function relationships, for the isolation of novel proteins involved in cytoskeletal function, and for deciphering the signals that regulate actin assembly in vivo. This review focuses on the budding yeast Saccharomyces cerevisiae and also identifies some recent advances from studies on the fission yeast Schizosaccharomyces pombe, for which studies on the actin cytoskeleton are still in their infancy.
Current
Introduction: cytoskeleton
the functions
Opinion
in Cell Biology
1994,
6:110-l
19
of the yeast actin
Although insights have been gained into the functions of the actin cytoskeleton of the budding yeast through the study of cytoskeletal mutants, its specific cellular functions remain elusive. The asymmetrical distribution of actin structures throughout the cell cycle (Fig. 1) suggests they have a role in directing new cell wall and membrane materials to growing regions of the cell. Cortical actin structures (referred to as patches) are located selectively at sites of surface growth: the bud, the septum [1,21 and the mating projection i3-61. Furthermore, cytoplasmic actin cables are oriented along the axes of formation of the bud [1,21 and the mating projection Ei-61.The phenotypes of cytoskeletal mutants (Table 1) indicate that the actin cytoskeleton functions in directing cell-surface growth. Mutations in the single essential actin gene (ACTI), and in other actin cytoskeletal genes, that disrupt the polarized organization of cortical actin structures result in an inability to undergo apical growth. The mechanisms through which actin participates in polarized cell-surface growth are not known. Defects in secretion, apparently in the late stages of the pathway (Golgi to plasma membrane) [71, are exhibited by act1 mutants, suggesting that actin might function in the movement of secretory vesicles. A direct role for actin in the movement of vesicles has yet ‘to be demonstrated. A&n-binding proteins that might mediate interactions between actin and secretory vesicles include Myo2p, a member of the dilute family of myosin I (unconventional or mini-myosin) proteins [81 that are thought to mediate vesicle movement along actin filaments, and tropomyosin, which regulates the
0
Actin,
fimbrin,
tropomyosin
Actin,
Abplp,
capping
I
protein,
cofilin,
fimbrin
0 1994 m Currm
Fig. 1. Organization
cerevisiae actin
and protein cytoskeleton.
components
Opinion in Cell Biolow
of the
Saccharomyces
interaction between myosin and actin in muscle cells and stabilizes actin cables in yeast cells [91. Both my02 [lOI and tpml (tropomyosin) ill’1 mutants accumulate membrane vesicles; however, defects in secretion are either not observed or are subtle. Identification of the origin of the membrane vesicles that accumulate in the mutants may help to further elucidate the roles of the MY02 and PM1 gene products in membrane trafficking. Recently, an important insight has been gained into one cellular function of actin. Kubler and Riezman [12*1 showed that actin and the actin-binding protein
Abbreviations ABP-actin binding protein; ACT-actin; ANC-actin RAH-reversion of act1 high osmolarity sensitivity; SLA-synthetic lethal with ABPl; SK-synthetic
110
0 Current
non-complementing; CAP-capping; SAC-suppressor of act1 temperature lethal with cap2; WY-suppressor
Biology
Ltd ISSN 0955-0674
COF-cofilin; MYO-myosin; sensitivity; SH-Src homology; of myosin; tpm-tropomysin.
The yeast actin cytoskeleton
‘abk
la. Genes involved
in function
of the hcchammyces
HOW
NUN
Cellular defects
Subcellular
lame
identitied
phenotype
in mutants*
localization
UPI
Biochemistry
Sequence
Holtzman
and Drubin
cerevisiae actin cyroskeleton.
;ene
CT1
Welch,
None detected
Lethal
homology
Cytoskeleton*” (Abpl p overproduction)
Actin patches
Morphology, cyioskeleton, secretion,
Cables and patches
Sequence similarities
References**
SH3 fsrc homology 3) domain, cofilinlike domain
I1 2’,36’,39*, 48.,771
Actin
osmoregulation, endocytosis, vesicle accumulation 4cr.7
sequence
lethal
n.d.
Actin-related
n.d.
proteins
1521
homology Genetics
tNC2
Genetics
n.d.
n.d.
n.d.
n.d.
136%39*1
tNC3
Genetics
n.d.
n.d.
n.d.
n.d.
136’,39’1
tNC4
Genetics
n.d.
n.d.
n.d.
n.d.
136’,39.1
3Pl
Biochemistry
ZAP2
Sequence homology
XT1
Biochemistry
WY01
Biochemistry
WY02
Genetics
Temperature sensitive
Morphology, Woskeleton, osmoregulation
Nucleus
4NCl
Morphology, cytoskeleton
Morphology. cytoskeleton
Actin patches
Morphology, cytoskeleton
Morphology, cytoskeleton
Actin patches
lethal
Cytokinesis, cell wall, osmoregulation*‘*’ Lethal
n.d.
Cytokinesis. cell wall, osmoregulation Morphology, cytoskeleton,
Human Enl and AF-9 proteins (implicated in acute leukemias)
Mammalian
capZ
[36*,39*]
129.,31,48*1
a-subunit
Aclin patches
n.d.““’
n.d.
Mamalian capZ bsubunit
129’,30,31, 42’,48’1
Low molecular weight severing proleins cofilin. actophorin, depactin, destrin
[27*,281
Myosin II (muscle myosin) heavy chain
I81
Myosin I (dilute family)
l8.10.11~ 58’1
vesicle accumulation
‘Not all phenotypes were evaluated for all mutants. **All references to each gene cited in the text are listed. For additional references, see literature cited within a given ,eference. References for ACT1 are too numerous IO list here. “‘The cellular defects were observed in cells with mutations in the indicated genes except in the case of the 48PJ gene, where the defects were seen when Abpl p was overproduced. *“The phenotype of a disruption mutation that removes the entire coding region has not been deponed. ““‘Although My01 p was reported to be located at the bud neck, this result has been called into question due to the observation that many non-affinity-purified ,abbit antisera will stain the bud neck. n.d. means not determined.
fimbrin (encoded by the SACG gene; [131> play an essential role in the internalization step of receptormediated endocytosis. The involvement seems to be direct, and not due to general defects in the secretory pathway, because a set (secretion) mutant with secretory defects similar to those of act1 mutants does not exhibit defects in endocytosis. Actin has also been shown to participate in endocytosis in epithelial cells [141. The powerful genetics available in yeast present a unique opportunity to study the role of the actin cytoskeleton in endocytosis in tivo. This review focuses on advances made in recent investigations into the cytoskeletal components of S. cerevisiae and S. pombe.
Actin structure/function S. cerevisiae provides two main advantages for the study of structure/function questions pertaining to actin. Firstly, a single, essential gene (AC73 encodes the sole source of ‘conventional’ actin in the cell (see also the section on actin-related proteins), thus eliminating concerns about isotype-specific functions and permitting the purification of biochemical quantities of a specific mutant actin. Yeast actin is 88% identical at the amino acid level to rabbit muscle actin [15,161, and is biochemically similar to it [17,18,19’1, suggesting that what is learned from studies in yeast will be generally
111
112
Cvtoskeleton
applicable. Secondly, homologous recombination can be employed to introduce the mutated version of the actin gene back into its chromosomal locus to assess its ability to function in viva. Several recent studies have used site-directed mutagenesis to address questions concerning actin structure and function in vivo and in vitro. One approach has been to target residues that have been previously implicated, either through biochemical crosslinking studies or studies on the crystal struchbk Gene name
PFYl
lb. Genes involved
in function of the Saccharomyces
HOW identified
Biochemistry, sequence homology
ture of monomeric actin, in specific aspects of actin function [19’,20,21*1. Rubenstein and colleagues have demonstrated the importance of the amino-terminal acidic residues of yeast actin for the interaction with myosin [19',21'1. In another study, Chen et al. 122'1 have examined the importance of the ‘hydrophobic plug’ in stabilizing actin filaments. According to the model proposed by Holmes et al. 1231 for filamentous actin, the hydrophobic plug accounts for the main interstrand contacts along the two-start helix (i.e two right-handed, intertwined helices). When a
cerevisiae actin cytoskeleton.
NUll
Cellular defects
Subcellular
phenotype
in mutants*
localization
very sick
Morphology, cytoskeleton
Diffuse cytoplasmic
Sequence similarities
Actin monomer binding protein
References*’
[49’.5cr,5
profilin
RAHl
Genetics
n.d.
nd.
n.d.
n.d.
132.1
RAHZ
Genetics
n.d.
r-cd.
n.d.
n.d.
1321
RAH3
Genetics
n.d.
n.d.
n.d.
132.1
5ACl
Genetics
Cold sensitive
Cytoskeleton Cytoskeleton, inositol auxotrophy
SAC2
Colgi and endoplasmic
No significant similarities
133.43.44’1
reticulum
n.d.
Cytoskeleton, secretion
n.d.
n.d.
1331
Cytoskeleton
n.d.
nd.
I331
SAC3
Genetics
n.d.
SAC4
Genetics
n.d.
n.d.
n.d.
n.d.
1331
SACS
Genetics
n.d.
n.d.
n.d.
n.d.
1331
SAC6
Genetics and biochemistry
Temperature sensitive
Morphology, cytoskeleton, endocytosis
Actin cables and patches
SAC7
Cold sensitive
Cytoskeleton
n.d.
SLA I
Temperature sensitive
Morphology, cytoskeleton
n.d.
Temperature sensitive
Morphology, cytoskeleton
n.d.
Actin filament bundling protein fimbrin No significant similarities St13 lsrc homology
II 2’.13,34. 36’,39’,40’ 42*.48-l I351
I4W
3) domains, sperm protein bindin
SLAZ
Genetics
SLCl
Genetics
n.d.
Morphology, cyloskeleton
n.d.
n.d.
142-l
SLCZ
Genetics
n.d.
Morphology, cytoskeleton
n.d.
n.d.
142.1
lPM1
Biochemistry
II
Temperature sensitive
Morphology, cytoskeleton, vesicle
Actin cables
Talin fcarboxyl terminal 200 amino acids)
Tropomyosin
140.I
[9,11’,36’, 390, 46’1
accumulation ‘Not all phenotypes were evaluated for all mutants. **All references to each gene cited in the text are listed. For additional references, see literature cited within a given reference. References for ACT1 are too numerous to list here. “‘The cellular defects were observed in cells with mutations in the indicated genes except in the case of the ABPt gene, where the defects were seen when Abplp was overproduced. “*CThe phenotype of a disruption mutation that removes the entire coding region has not been rewed. *****Although Myolp was reported to be located at the bud neck, this result has been called into question due to the observation that many non-affinity-purified rabbit antisera will stain the bud neck. n.d. means not determined.
The yeast actin cytoskeleton
charged residue (Let1266+A~p) was introduced into this region [22*1, yeast depending on this mutant actin showed a modest cold-sensitive growth defect. Significantly, in vitro studies showed that this mutation creates a change in the assembly properties of the actin monomer, including cold-sensitive defects in nucleation and filament elongation. An alternative approach to study structure/function relationships was carried out by Wertman et al. 124’1. Mutations were targeted to the surface of the protein by identifying clusters of two or more charged residues in the primary sequence; every charged residue in a cluster was replaced by alanine. The majority of the altered residues (81 o/o>are at or near the surface of the protein and are therefore unlikely to alter the tertiary structure of the protein. The resulting mutant actins were assayed in vivo by gene replacement. Sixteen conditional-lethal alleles were isolated, and many mutations corroborated predictions of the atomic model for actin filaments [24*,25,26’1. In addition, this collection has proven useful in dissecting the in vivo functions and biochemical properties of actin. For example, several mutations interfere with the organization of mitochondria in the cell, and another causes a defect in the interaction between actin filaments and the mushroom toxin phalloidin 126’1. Further study of actin mutants in budding yeast should result in additional insights into the relationship between actin structure and actin function.
Identification
of actin-binding
proteins
The functions of actin rely on the activity of a variety of actin-binding proteins. Although yeast cells do not exhibit behaviors such as motility that might necessitate a high degree of cytoskeletal complexity, it has become apparent that yeast is typical of eukaryotes in its complement of actin-binding proteins. Most classes of actin-binding proteins found in other eukaryotes have been identified in yeast (Table 1). Recently, Moon et al. 127’1 have identified a yeast homologue of the low molecular weight actin filament severing protein, cofilin, which is encoded by the COFl gene (this gene was also identified by Iida et al. 1281). Cofilin is an essential protein located at cortical actin structures. In addition, Moon et al. have identified three more activities in yeast extracts that may represent other actinbinding proteins 127’1.
et al. 1291 have identified a homologue of the a-subunit of vertebrate capping protein (encoded by the CAP1 gene). Capping protein functions as a heterodimer of one a-subunit and one p-subunit (encoded by CAp2, previously identified in 13011,and is located at cortical actin structures 1311.Deletion of one or both of the genes encoding the a- and p-subunits causes aberrant cell morphology, disorganization of cortical actin structures and partial disappearance of cytoplasmic actin cables 129’,3Ol. Therefore, capping protein might function to nucleate or stabilize actin cables and other
Amatruda
Welch,
Holtzman
and Drubin
actin filament structures in vivo, a role that would be consistent with its biochemical activity in vitro.
Genetic identification cytoskeletal function
of proteins
important
for
Genetic techniques have been used to identify a variety of additional genes that are important for actin cytoskeletal function in yeast. The significance of using novel genetic approaches is underscored by the observation that many of these genes have not been identified through biochemical methods in other organisms. Using mutant alleles of the ACTI gene, two novel avenues were taken to identify genes important for actin function. Chowdhury et al. 132’1 selected for mutations that suppress a specific phenotype of act1 mutants, sensitivity to high osmolarity (osmosensitivity). Previously, mutations in the SACl-SAC7 genes were identified as suppressors of the temperature sensitivity of act1 mutants 133351. Chowdhury et al. identified three genes (IUHl, RAH2 and RAH.. reversion of act1 high osmolarity sensitivity), one of which (Z?&f..) is known not to be allelic with any previously identified cytoskeletal gene. Mutations in &I./Y3 cause defects in the organization of cortical actin structures, indicating that Rah3p, its protein product, is important for actin cytoskeletal function. In another study, Welch et al. 136’1 screened for extragenic mutations that fail to complement the temperature-sensitive phenotype exhibited by act1 alleles, a technique that has been used to identify genes that encode physically interacting proteins 137,381. At least four new genes, ANCl-ANC4 (actin non-complementing), were identified. Mutations in these genes interact genetically with mutations in genes for a&-i-binding proteins 139’1, providing evidence that the proteins participate in cytoskeletal function. Furthermore, mutations in AhCl cause defects in actin organization, suggesting that Anclp is important for cytoskeletal function 136’1. Another approach to identify cytoskeletal genes has been screening for mutations that exhibit specific genetic interactions with alleles of genes encoding actinbinding proteins. Holtzman et al. 140’1 identified SLAl and SLA2 (synthetic lethal with a&l) (and rediscovered SACG, which encodes the actin-binding protein fimbrin 1131) by screening for mutations that are lethal in combination with a null mutation in ABPl (actinbinding protein; Table la>. Slalp, like Abplp, has SH3 domains (SIC homology domain 31, which are present in other membrane cytoskeletal proteins and may link the cytoskeleton to intracellular signaling pathways 1411.Sla2p has a 200-amino-acid carboxy-terminal domain that is similar to the carboxy terminus of talin, a component of Iibroblast focal adhesions. Mutations in both X41 and SLA2 cause unique defects in the organization of cortical actin structures, indicating that each protein plays a different role in the
113
114
Cvtoskeleton
functions of the cortical cytoskeleton. Using a similar approach, Karpova et al. [42*1 isolated mutations in SACG and in two additional genes (SLCl and SLCZ synthetic lethality with cap2) by screening for mutations that are lethal in combination with a null mutation in the CAP2 gene. Defects in cytoskeletal organization are exhibited by slcl and slc2 mutants, indicating that Slclp and Slc2p are involved in actin function. It is now important to determine the intracellular location of the RAHl-3 ANCl-4, $31-2, SLCl-2 gene Jducts, and the molecular nature of the interaction between these proteins and actin. Genetic approaches have sometimes led to the identification of genes whose functional relationship to actin remains difficult to elucidate, although this does not diminish the importance of the relationship. Mutations in SAC1 were identified as suppressors of act1 mutations 1331, and were subsequently found to suppress defects caused by loss of the phosphatidylinositol/phosphatidylcholine transfer protein, Secl4p [431, suggesting that Saclp has a function in secretion and/or membrane metabolism. Whitters et al. [44'1 have recently demonstrated that Saclp is an integral membrane protein located in the endoplasmic reticulum and Golgi, and that sac1 mutants have an unusual inositol auxotrophy. These results suggest that Saclp is likely to function in inositol glycerophospholipid metabolism, and raise the question of the mechanisms through which actin and Saclp interact. One potential mechanism is through a&n-binding proteins whose activities may be regulated by phospholipids. A connection between phospholipid metabolism and actin cytoskeletal function has been suggested by biochemical studies 145-471. Genetic analysis provides one avenue towards testing the in z&o relevance of these interactions.
Genetic function
analysis of a&n-binding
protein
Actin-binding proteins with a variety of biochemical activities must act together to produce a functional cytoskeleton. Issues such as whether certain subgroups of actin-binding proteins work together in tivo can be addressed by identifying genetic interactions between mutations in different genes coding for actin-binding proteins. To this end, several groups l11’,39’,40’,42*,48*1 have examined the consequences of making combinations of null mutations in genes that encode the yeast actin-binding proteins Abplp, fimbrin, tropomyosin, capping protein and Myo2p, as well as in other genes implicated in cytoskeletal function. Specific pair-wise combinations of null mutations resulted in lethality. Surprisingly, none of these pairs of genes encode proteins with similarity in structure or biochemical activity. This suggests that although these proteins may have functional homologies, their effects on the cytoskeleton are achieved through different mechanisms.
Genetic studies have also addressed the functions of the actin monomer binding protein profilin. It has been suggested that this protein participates in multiple (and perhaps related) processes, including actin cytoskeletal function and PIP2 (phosphatidylinositol 4, 5bisphosphate)-mediated signaling [46,471, yet its role in vivo remains uncertain. A report by Magdolen et al. [49*1 showing that overproduction of profilin can suppress the lethality caused by overproduction of actin suggests that, at least under certain conditions, profilin may act in vivo to sequester monomeric actin. A promising avenue for the elucidation of profilin function is the creation of site-directed mutations that affect specific activities of profilin. Using this approach, Haarer et al. 150’1 have created mutations that affect actin and/or PIP2 binding. Analysis of these mutants suggests that the actin-binding property of profilin is important for its in vivo function. Questions regarding the relevance of PIP2 binding in vivo remain unresolved (see also 1511).
Actin-related interactions
proteins
and actin-microtubule
In general, actins from different species are greater than 70% identical in amino acid sequence, with the primary amino .acid sequence of S. cerevisiae actin being 88% identical to mammalian actins. Interestingly, a family of actin-related proteins that are 4+50% identical to actin in their amino acid sequences have been discovered in S. cerevisiae (AcQp 15211,Schizosaccbaromyces pombe (act2p 1531) and in mammalian cells (actin-RPV [541, centractin [55,561, actin [571). The S. cerevisiae ACT2 and S. pombe act2 genes are essential, but their cellular functions have yet to be determined. Vertebrate actin-related proteins are involved in microtubule-based vesicle motility 154-571, and by analogy, the yeast proteins may perform similar functions. Determining the cellular location of Act2p and identifying proteins that interact biochemically and genetically with Act2p in yeast will increase our understanding of the functions of actin-related proteins in eukaryotic cells. Another instance of possible convergence between the functions of actin and microtubules is the finding by Lillie and Brown 158’1 that overproduction of Smylp @MYI = suppressor of myosin), which shares sequence similarity with the motor domain of the microtubule-based motor protein kinesin, can suppress mutations in the MY02 gene, which encodes a myosin I actin filament-based motor protein. Although deletion of SMYl causes no detectable phenotype, the deletion is lethal in combination with a MY02 mutation, providing more evidence that Smylp and Myo2p proteins perform similar functions in vivo. The sequence similarities to motor proteins suggest that Smylp and Myo2p might be important for vesicle transport, and present the interesting possibility that the actin and mi-
The yeast actin cytoskeleton Welch, crotubule cytoskeletons in this respect.
perform
redundant
functions
Another cellular process in which both actin and microtubules participate is the alignment of the mitotic spindle. Before anaphase, the mitotic spindle is positioned with one spindle pole at or through the mother-bud neck, allowing proper chromosome segregation during anaphase. Palmer et al. WI showed that spindles are misoriented in act1 mutants and in mutants with disrupted astral microtubules, suggesting that astral microtubules interact with the actin cytoskeleton to orient the spindle. This is consistent with the finding that pharmacological disruption of the actin cytoskeleton in Caenorhabditis elegans blastomeres changes the position of the spindle 160,611, and that actin function is necessary for migration of the nucleus to the tip of the mating projection in S. cerevisiue [61.
Signaling/cell
cycle control
of actin organization
The asymmetric disposition of actin in S. cerevisiae is one cellular readout of complex genetic pathways that influence morphogenesis, bud site selection and the formation of the pheromone-induced mating projections. (For a comprehensive review of the genetic control of cell polarity in Succbaromyces, see 162X341). Several recent papers have begun to explore the signaling pathways that control the rearrangements of the yeast actin cytoskeleton. Given the rapid cytoskeletal reorganizations that are possible in a variety of cell types (1651 and references therein), it is certain that post-translational modifications and direct modulations by second messenger molecules are central to the regulation of actin assembly in vivo. Using a variety of mutants and misexpression strategies, Lew and Reed 16@1have addressed the role of the Cdc28 kinase in the regulation of morphogenesis and the localization of cortical actin structures throughout the cell cycle. Importantly, they find that alterations in the expression of Gl and G2 cyclins have dramatic effects on polarization of the cytoskeleton. In addition, reorganization of actin following the downshift of a temperature-sensitive cdc28 mutation was found to be independent of de novo protein synthesis, suggesting that phosphorylation plays a direct role in the regulation of the yeast actin cytoskeleton. It is now important to determine whether any cytoskeletal components in yeast are direct targets for the Cdc28 kinase. Calcium has well documented effects on the activity of several actin-binding proteins isolated from motile cells (see 1651 for examples), and it is a common second messenger molecule generated in response to diverse cell-surface signals. Yeast contain a single essential gene (CMDI) that encodes calmodulin, a high affinity calcium-binding protein implicated in diverse cellular functions 167,681. Calmodulin has now been immunolocalized in S. cerevisiae and found associ-
Holtzman
and Drubin
ated with sites of surface growth throughout the cell cycle 169’,701, with the pattern of calmodulin localization that is overlapping but distinct from that of actin. Interestingly, mutations in calmodulin can affect the localization of actin 163’1 (Y Ohya, personal communication) suggesting that calcium signaling mechanisms may function in the polarization of the yeast actin cytoskeleton.
CDC42, a gene involved in development
of cell polarity and asymmetric assembly of the actin cytoskeleton in S. cerevisiue, is a member of the rho family of ras-related small GTP-binding proteins 171,721. Matsui and Toh-e have now identified two new members of this family, RHO3 and RHO4 1731, and have demonstrated their involvement in development of cell polarity 174.1. Strains lacking RHO3 and RHO4 have defects in morphogenesis and delocalized actin cytoskeletons. Additionally, Chenevert et al. 1751find that the growth defects of the rho.3 strain can be suppressed by overexpression of either CDC42 or BEMl, an SHIcontaining gene involved in polarity development . The identification in mammalian cells of an SH3 ligand with homology to rho-GTPase activator proteins 1761has generated interest in the link between yeast SH3 proteins (Abplp, Bemlp, Slalp and Rvs167p; 140’,75,77,78*1) implicated in morphogenesis and actin cytoskeleton organization, and the other proteins that control cell polarity. In addition, the recent discovery that rho proteins control actin stress fiber assembly 1791 in Iibroblasts highlights the emerging parallels between the regulation of the actin cytoskeleton in yeast and in more complex cells.
Schizosaccharomyces
pombe
While studies on the actin cytoskeleton in fission yeast are in their infancy compared with those in budding yeast, similar advantages are afforded by S. pombe for addressing the function of the actin cytoskeleton. It will be useful to compare and contrast cytoskeletal regulation in these two yeasts as a way to identify conserved mechanisms important for cytoskeleton function. S. pombe is a particularly promising organism for elucidation of the mechanism and regulation of cytokinesis, because cytokinesis appears more similar to the process in vertebrate cells 180,811, because a large number of mutants defective in cytokinesis are available 1821, and because cell-cycle regulation in S. pombe is well understood 1831. Balasubramaman et al. t84.1 have now shown that the cdc8 gene of S. pombe encodes a novel tropomyosin essential for cytokinesis, and have localized this tropomyosin to small actin patches as well as to the medial band of filamentous actin that forms at the septum. In addition, antibody cross-reactivity was found in S. pombe against transgelin, a widely distributed actin filament gelation factor which is downmodulated in mammalian cells by transformation and non-adherent culture conditions 185’1.
115
116
Cvtoskeleton
.
Conclusions
The past year and a half has seen a sharp rise in activity in research on the yeast actin cytoskeleton. Many new genes have been identified and their in vivo roles tested, and the potential for identifying regulatory interactions and structure-function relationships is being realized. Because a concerted effort by a large research community is being focused on understanding virtually every aspect of the biology of the simple yeast cell, we can look forward to the development of an integrated view of cytoskeletal function and regulation.
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and recommended
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24. .
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27. .
40. .
39. .
26. .
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33.
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42. .
Mutations that EnIn Saccbarumyces Morphogcncsis, I35:693709. screen for mutations the capping protein were shown to be
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44. .
WHI~RS EA. CLEVES AE. MCGEE Tp, SKINNER HB, BANKAIIX VA: SAClp is an Integral Membrane Protein that Influences the Cellular Requirement for Phospholipid Transfer Protein Function and lnositol in Yeast. J Cell Bfol 1993, 122:79-94. Saclp, originally identified in a screen for suppressors of an actin mutation, is shown to be an integral membrane protein, resident in the endoplasmic reticulum and Golgi. Consistent with previous findings that Saclp functionally interacts with a phospholipid transfer protein (Secl4p), evidence is presented that Saclp is likely to function in inositol glycerophospholipid metabolism. 45.
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38.
41.
Yeast Cener-
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MD Welch, DA Holtzman and DG Drubin, Department of Molecular and Cell Biology, 455 Life Sciences Addition, University of California, Berkeley, CA 94720, USA.
Author for correspondence:
DG Drubin.
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