Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane. This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus. We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes. Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane. We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface.

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin–binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

The human cytomegalovirus tegument protein, pTRS1, appears to function at several discrete stages of the virus replication cycle. We previously demonstrated that pTRS1 acts during the late phase of infection to facilitate the production of infectious virions. We now have more precisely identified the late pTRS1 function by further study of a mutant virus lacking the TRS1 region, ADsubTRS1. We observed a significant reduction in the production of capsids, especially DNA-containing C-capsids, in mutant virus-infected cells. ADsubTRS1 exhibited normal cleavage of DNA concatemers, so the defect in C-capsid production must occur after DNA cleavage and before DNA is stably inserted into a capsid. Further, the normal virus-induced morphological reorganization of the nucleus did not occur after infection with the pTRS1-deficient mutant.Cytomegalovirus, a member of the betaherpesvirus subfamily, is a large DNA virus with a narrow host range (19). Human cytomegalovirus (HCMV) infections are widespread and generally asymptomatic in healthy individuals. After infection, the virus can persist in a lifelong, latent state. Although relatively little is known about the events involved the establishment of HCMV latency, the cascade of gene expression and the events that occur during the lytic replication cycle of the virus have been studied in considerable detail (24).The genome of HCMV contains two unique domains, one long and one short, each flanked by repeat sequences. Two open reading frames, TRS1 and IRS1, include sequence from both repeated and unique domains. The N-terminal two-thirds of pTRS1 is encoded in the c repeat region, and the remainder of the protein is coded within the unique short region. The related protein, pIRS1, is encoded in the internal or cЈ repeat region plus the adjacent unique short region. Consequently, the N-terminal domains of pTRS1 and pIRS1 are nearly identical, and the two proteins have different C-terminal domains (37). Because their amino-terminal domains are encoded in the repeat region, the transcription of these genes is controlled by identical immediate-early promoters. Both pTRS1 and pIRS1 are packaged into the virion and therefore are delivered to the cell immediately upon infection (32); pTRS1 and pIRS1 can be detected in infected cells as early as 2 h postinfection (33, 36).The first function ascribed to pTRS1 and pIRS1 was transcriptional activation. Both proteins were found to act in conjunction with the immediate-early transcriptional regulatory proteins, IE1 and IE2, but not on their own, to increase expression from the UL44 promoter in transient-transfection assays (36). Subsequent analysis identified TRS1/IRS1 as 1 of 11 loci that are required for transient complementation of HCMV oriLyt-dependent DNA replication (28). In this assay, pTRS1 and pIRS1 likely facilitate the accumulation of other proteins that function directly in the replication process (15). More recently, pTRS1 and pIRS1 have been shown to block the double-stranded RNA-dependent protein kinase R response pathway,...