There is a debate over how protein trafficking is performed through the Golgi apparatus. In the secretory pathway, secretory proteins that are synthesized in the endoplasmic reticulum enter the early compartment of the Golgi apparatus called cis cisternae, undergo various modifications and processing, and then leave for the plasma membrane from the late (trans) cisternae. The cargo proteins must traverse the Golgi apparatus in the cis-to-trans direction. Two typical models propose either vesicular transport or cisternal progression and maturation for this process. The vesicular transport model predicts that Golgi cisternae are distinct stable compartments connected by vesicular traffic, whereas the cisternal maturation model predicts that cisternae are transient structures that form de novo, mature from cis to trans, and then dissipate. Technical progress in live-cell imaging has long been awaited to address this problem. Here we show, by the use of high-speed three-dimensional confocal microscopy, that yeast Golgi cisternae do change the distribution of resident membrane proteins from the cis nature to the trans over time, as proposed by the maturation model, in a very dynamic way.

SummaryProtein export from the endoplasmic reticulum (ER) to the Golgi apparatus occurs at specialized regions known as the ER exit sites (ERES). In Saccharomyces cerevisiae, ERES appear as numerous scattered puncta throughout the ER. We examined ERES within the peripheral ER, finding that the proteins comprising the ERES localize on high-curvature ER domains where curvature-stabilizing protein Rtn1 is present. Drtn1 Drtn2 Dyop1 cells have fewer high-curvature ER domains, but ERES accumulate at the remaining high-curvature ER domains on the edge of expanded ER sheets. We propose that membrane curvature is a key geometric feature for the regulation of ERES localization. We also investigated a spatial relationship between ERES and Golgi cisternae. Golgi cisternae in S. cerevisiae are unstacked, dispersed, and moving in the cytoplasm with cis-cisternae positioned adjacent to ERES, whereas trans-cisternae are not. Morphological changes in the ER of Drtn1 Drtn2 Dyop1 cells resulted in aberrant Golgi structures, including cis-and trans-markers, and there was reduced movement at ERES between expanded ER sheets and the plasma membrane.

During plant reproduction, sperm cells are delivered to ovules through growing pollen tubes. This process involves tip-localized receptor kinases regulating integrity and/or guidance of pollen tubes, whose localizations must be strictly regulated. However, the molecular basis for tip-localization of these molecules remains largely elusive. Here we show that a pair of AP180 N-terminal homology domain-containing proteins, PICALM5a and PICALM5b, is responsible for the tip-localization of ANXUR receptor kinases acting in an autocrine signaling pathway required for pollen tube integrity in Arabidopsis thaliana. The picalm5a picalm5b double mutant exhibits reduced fertility, and the double mutant pollen is defective in pollen tube integrity with premature bursts. The tip localization of ANXUR proteins is severely impaired in picalm5a picalm5b pollen tubes, whereas another receptor kinase PRK6 acting in pollen tube guidance is not affected. Based on these results, we propose that PICALM5 proteins serve as specific loading adaptors to recycle ANXUR proteins.

One table, seven figures, and two supplemental figures. 27 Word count: 3,967 words 28 29 Running title: KAKU4 controls migration of the nucleus in pollen tubes 30 2 Highlight 31The nuclear envelope protein KAKU4 is involved in controlling the migration order of 32 vegetative nuclei and sperm cells in pollen tubes, affecting the competitive ability of 33 pollen for fertilization. 34 35 36 Abstract 37 A putative nuclear lamina protein, KAKU4, modulates nuclear morphology in 38 Arabidopsis thaliana seedlings but its physiological significance is unknown. KAKU4 39 was strongly expressed in mature pollen grains, each of which has a vegetative cell 40 and two sperm cells. KAKU4 protein was highly abundant on the envelopes of 41 vegetative nuclei (VNs) and less abundant on the envelopes of sperm cell nuclei 42 (SCNs) in pollen grains and elongating pollen tubes. VN is irregularly shaped in 43wild-type pollen. However, KAKU4 deficiency caused it to become more spherical. 44 These results suggest that the dense accumulation of KAKU4 is responsible for the 45 irregular shape of the VNs. After a pollen grain germinates, the VN and SCNs migrate 46 to the tip of the pollen tube. In the wild type, the VN preceded the SCNs in 91-93% of 47 the pollen tubes, whereas in kaku4 mutants, the VN trailed the SCNs in 39-58% of the 48 pollen tubes. kaku4 pollen was less competitive than wild-type pollen after pollination, 49 although it had an ability to fertilize. Taken together, our results suggest that 50 controlling the nuclear shape in vegetative cells of pollen grains by KAKU4 ensures 51 the orderly migration of the VN and sperm cells in pollen tubes. 52 53